JP2002356439A - Use of galanin-like peptide - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a new use of galanin-like peptide(GALP). SOLUTION: The invention relates to an agent for controlling the secretion of luteotropic hormone(LH), etc., containing galanin-like peptide(GALP), etc. The LH secretion controlling agent is useful as a preventing and treating agent for infertility, menstrual disorder, mesopausal disorder, dyspituitarism, obesity, etc.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to the use of a bioactive peptide. More specifically, the present invention relates to a ligand polypeptide for a G protein-coupled receptor (sometimes called a receptor) protein or a salt thereof, or a luteinizing hormone containing a DNA encoding the polypeptide (leutinizing hormone; LH) secretion regulator and luteinizing hormone-releasing hormone (leutin)
The present invention relates to an agent for regulating secretion of releasing hormone (LHRH).

[0002]

2. Description of the Related Art Many hormones and neurotransmitters regulate biological functions through specific receptors present on cell membranes. Many of these receptors are coupled to guanine nucleotide binding proteins (guanine nucleonucleotides).
Intracellular signal transduction is performed through activation of tide-binding protein (hereinafter sometimes abbreviated as G protein). Recently, we have discovered that the ligand among these G protein-coupled receptors, the so-called orphan G protein-coupled receptor, is a galanin receptor subtype, GA
LR2 [Journal of Biological Chemistry (J. Biol. Chem.), Vol. 272, p. 24612 (1997),
A peptide ligand for the FEBS Letter, Vol. 471, p. 225 (1997)] was discovered in the swine hypothalamus [Journal of Biological Chemistry (J. Biol. Che).
m.), 274, 37041 (1999)]. This peptide has a consensus sequence of 13 amino acids with galanin, so that it has a consensus sequence of galanin-like peptide (GALin-like Peptide (GAL
P)), but it is not known at all that GALP has an LH secretion regulating action or an LHRH secretion regulating action.

[0003]

As described above, GALP has a consensus sequence of 13 amino acids with galanin, but is expected to find a new physiological action different from galanin and to develop a useful medicine.

[0004]

Means for Solving the Problems The present inventors have conducted intensive studies in order to solve the above-mentioned problems, and as a result, intracerebroventricular administration of GALP and galanin to rats to examine changes in blood pituitary hormone levels. did. As a result, in the GALP administration group (5 nmol / rat), the blood luteinizing hormone (LH) concentration increased, and no effect on other pituitary hormones was observed. In addition, blood LH observed in GALP
No effect of increasing the concentration was seen with galanin. Current,
There is no known peptidic factor that causes an increase in blood LH concentration by intraventricular administration. In addition, GALP was observed in Zucker fatty rats with abnormal leptin receptors.
Showed a significant increase in blood LH at 1 nmol / rat, but no change in blood LH concentration was observed in Zucker lean rats, which were control animals, and GALP reactivity was improved in Zucker fatty rats. . The present inventors have further studied based on these findings, and as a result, have completed the present invention. That is, the present invention relates to (1) a luteinizing hormone (LH) secretion regulator containing GALP or a salt thereof, or a DNA encoding GALP; (2) GALP having the same amino acid sequence as SEQ ID NO: 35 or The LH secretion regulator according to the above (1), which is a peptide containing substantially the same amino acid sequence; (3) GALP is SEQ ID NO: 31, 33, 34 or 6
LH secretion regulator according to the above (1), which is a peptide containing the same or substantially the same amino acid sequence as the amino acid sequence represented by 2; (4) the DNA encoding GALP is SEQ ID NO: 32;
The LH secretion regulator according to the above (1), which is a DNA containing a nucleotide sequence that hybridizes under high stringent conditions with the nucleotide sequence represented by 39, 40 or 64; (6) LH secretion regulator according to (1), which is an LH secretion inhibitor; (7) infertility, irregular menstruation, dysmenorrhea, amenorrhea, rare occurrence The LH secretagogue according to the above (5), which is a preventive / therapeutic agent for menstruation, premenstrual syndrome, menopause, pituitary dysfunction or obesity; (8) prostate cancer, benign prostatic hyperplasia, precocious puberty or L
The LH secretion inhibitor according to the above (6), which is a prophylactic / therapeutic agent for H-producing pituitary tumor; (9) LH secretion regulator containing a GALP agonist; (10) LH secretion containing a GALP inhibitor (11) a luteinizing hormone-releasing hormone (LHRH) secretion regulator containing GALP or a salt thereof, or DNA encoding GALP; (12) GALP identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 35 The LHRH secretion regulator according to the above (11), which is a peptide containing the same amino acid sequence; (13) GALP is identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 31, 33, 34 or 62 The LHRH secretion regulator according to the above (11), which is a peptide having the same amino acid sequence; (14) DNA encoding GALP Is SEQ ID NO: 3.
The LHRH secretion regulator according to the above (11), which is a DNA containing a nucleotide sequence that hybridizes under high stringent conditions to the nucleotide sequence represented by 2, 39, 40 or 64; (15) an LHRH secretion promoter (16) an LHRH secretion modulator according to (11), which is an LHRH secretion inhibitor; (17) infertility, irregular menstruation, dysmenorrhea, amenorrhea; The L according to the above (15), which is a preventive or therapeutic agent for rare menstruation, premenstrual syndrome, menopause, pituitary dysfunction or obesity.
(18) the above-mentioned (1) which is an agent for preventing or treating prostate cancer, benign prostatic hyperplasia, precocious puberty or LHRH-producing pituitary tumor;
(19) a LHRH secretion regulator containing a GALP agonist; (20) an LHRH secretion regulator containing a GALP inhibitor; (21) an LH secretion regulator or LHRH secretion regulator Use of GALP or a salt thereof, or DNA encoding GALP for the manufacture of an agent; and (22) a pharmacologically effective amount of GA for a mammal.
LP or its salt, or DN encoding GALP
The present invention relates to a method for preventing or treating LH secretion disorder or LHRH secretion disorder, which comprises administering A.

[0005]

BEST MODE FOR CARRYING OUT THE INVENTION In the present specification, “substantially the same” refers to the activity of a protein, for example, LH secretion regulating activity (action), LHRH secretion regulating activity (action), agonist activity on a receptor, that is, ligand activity. The activity of activating the receptor, the activity of binding the ligand to the receptor, and the like are substantially the same. Amino acid substitutions, deletions, additions or insertions often do not significantly alter the physiological or chemical properties of the peptide, in which case the substituted, deleted, added or inserted peptide is Would be substantially identical to those without such substitutions, deletions, additions or insertions. Substantially identical substitutions of amino acids in the amino acid sequence can be selected, for example, from other amino acids in the class to which the amino acid belongs. Non-polar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, methionine and the like. Glycine, serine, and polar (neutral) amino acids
Threonine, cysteine, tyrosine, asparagine, glutamine and the like. Examples of (basic) amino acids having a positive charge include arginine, lysine, histidine and the like. As negatively charged (acidic) amino acids,
Aspartic acid, glutamic acid and the like. GA
LP (hereinafter sometimes referred to as "the polypeptide of the present invention", and "GALP or a salt thereof" may be referred to as "the polypeptide of the present invention") has an ability to bind to a galanin receptor. Peptide. Preferably, it is a known ligand peptide other than galanin which has an activating effect on a galanin receptor. The galanin receptor is as described below.

The polypeptide of the present invention includes, for example,
An amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 35 (for example, SEQ ID NO: 1
3) and a peptide having LH secretion regulating activity (action) or LHRH secretion activity (action), specifically (I) identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 35 Amino acid sequence of (for example,
SEQ ID NO: 13, etc.) and SEQ ID NO: 11,
It contains an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 16 or SEQ ID NO: 43, and has LH secretion regulating activity (action) or LHRH secretion A peptide having activity (action), (II) having a molecular weight of 5,000 to 10,000
A peptide according to the above (I), which has a molecular weight of 5,000 to 8,000;
(IV) having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 17, and
Secretory regulation activity (action) or LHRH secretion activity (action)
And (V) the same or substantially the same as the amino acid sequence represented by SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 34 or SEQ ID NO: 62. Has an amino acid sequence,
And a peptide having LH secretion regulating activity (action) or LHRH secretion activity (action). further,
Examples of the polypeptide of the present invention include (VI) a peptide containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 36, (VI
I) SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 1
5, the peptide according to the above (VI), which contains the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 16 or SEQ ID NO: 43, (VIII)
The peptide according to (VII) having a molecular weight of 5,000 to 10,000, (IX) the peptide according to (VII), having a molecular weight of 5,000 to 8000, (X) SEQ ID NO:
31, a peptide containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 33, SEQ ID NO: 34 or SEQ ID NO: 62, (XI) amino acid sequence represented by SEQ ID NO: 17 A peptide containing the same or substantially the same amino acid sequence as (XI
I) containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 35 (for example, SEQ ID NO: 13 and the like), and having SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 15, A peptide containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 16 or SEQ ID NO: 43, (XII
I) The above (X) to above having a molecular weight of 5,000 to 10,000.
And the peptide described in (XII).

[0007] The polypeptide of the present invention, its production method and use are described in more detail below. Although the origin of the peptide of the present invention is not particularly limited, for example, warm-blooded animals (eg, human, guinea pig, rat, mouse, pig,
Tissues (eg, pituitary, pancreas, brain, kidney, liver, gonads, thyroid, gall bladder, bone marrow,
It may be a peptide derived from the adrenal gland, skin, muscle, lung, digestive tract, blood vessel, heart, testis, etc.) or cells (eg, mouse brain, rat brain, pig brain, bovine brain, pig hypothalamus, bovine Those derived from the hypothalamus, pig lung, bovine lung, bovine stomach, human hypothalamus, porcine testis, bovine testis, rat testis, mouse testis, human testis or human lung),
It may be a synthetic peptide. For example, polypeptides of the present invention include: SEQ ID NO: 17, SEQ ID NO: 31, SEQ ID NO: 33,
In addition to the peptide containing the amino acid sequence represented by SEQ ID NO: 34 or SEQ ID NO: 62 (preferably, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 34 or SEQ ID NO: 62), SEQ ID NO: 17, SEQ ID NO: 3
1, SEQ ID NO: 33, SEQ ID NO: 34 or SEQ ID NO:
62 (preferably, SEQ ID NO: 31, SEQ ID NO: 33,
About 50 to 99.9% (preferably 70 to 9) of the amino acid sequence represented by SEQ ID NO: 34 or SEQ ID NO: 62)
9.9%, more preferably 80-99.9%, even more preferably 90-99.9%, and most preferably 95-9%.
A peptide having an activity substantially equivalent to that of a peptide containing an amino acid sequence having 9.9% homology (provided that the sequence of galanin or its precursor represented by SEQ ID NO: 7, 8, 9, or 10) Ex).

"Substantially the same activity" includes, for example, LH secretion regulating activity (action), galanin receptor GA
An activity of binding to LR1, GALR2 or GALR3,
Galanin receptor GALR1, GALR2 or G
ALR3 activating activity or associated arachidonic acid release, intracellular Ca ²⁺ release, intracellular c
Inhibition of AMP production, inositol phosphate production (inhibition), fluctuation of cell membrane potential, phosphorylation of intracellular proteins, intracellular pH
Are homologous in their ability to activate signal transduction activity systems, such as alterations in the expression. Therefore, quantitative factors such as the intensity of these activities and the molecular weight of the peptide may be different. These activities can be measured by a known method or a method analogous thereto, for example, the methods described in Reference Examples 1, 17 and Examples 1 to 3 described below. Specific examples of the polypeptide of the present invention include the following (however, excluding galanin having the amino acid sequence represented by SEQ ID NO: 7, 8, 9, or 10 or a precursor thereof). (I) SEQ ID NO: 17, SEQ ID NO: 31, SEQ ID NO: 3
3, SEQ ID NO: 34 or SEQ ID NO: 62 (preferably, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 34
Or a mouse brain containing an amino acid sequence represented by the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 62) or a partial sequence thereof,
Rat brain, pig brain, bovine brain, pig hypothalamus, bovine hypothalamus, pig lung, bovine lung, bovine stomach, human hypothalamus, pig testis, bovine testis, rat testis, mouse testis, peptide from human testis or human lung . (II) SEQ ID NO: 17, SEQ ID NO: 31, SEQ ID NO:
33, SEQ ID NO: 34 or SEQ ID NO: 62 (preferably, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 34
Or a peptide containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 62) or a partial sequence thereof or a partial peptide thereof, wherein one or more amino acids are substituted, deleted, Peptides containing an added or inserted amino acid sequence are mentioned as peptides containing substantially the same amino acid sequence. For example, (1) SEQ ID NO: 17, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 34 or SEQ ID NO: 62 (preferably SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 34 or SEQ ID NO: : 62) or an amino acid sequence in which one or more and seven or less, preferably one or more and five or less, more preferably one or more and three or less amino acids are deleted in the amino acid sequence represented by or a partial sequence thereof, 2) SEQ ID NO: 17, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 34 or SEQ ID NO: 62 (preferably SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO:
34 or SEQ ID NO: 62) or 1 to 20 amino acids, preferably 1 to 15 amino acids, more preferably 1 to 10 amino acids are added to the amino acid sequence represented by SEQ ID NO: 62 or a partial sequence thereof (or 1 to 10 amino acids). (Inserted) amino acid sequence, (3) SEQ ID NO: 17, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 34 or SEQ ID NO: 62 (preferably SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: :
34 or SEQ ID NO: 62) or one or more and seven or less, preferably one or more and five or less, more preferably one or more and three or less amino acids in the amino acid sequence represented by And a peptide containing an amino acid sequence substituted with As the “substantially identical amino acid sequence”, 70 to 99.9%
More preferably 80 to 99.9%, even more preferably 9%
Amino acid sequences having 0-99.9%, most preferably 95-99.9% homology.

The molecular weight of the polypeptide of the present invention is about 5,000 when measured by gel filtration chromatography or the like.
From about 10,000 daltons, preferably from about 5000 to about 8
000 daltons, more preferably from about 5500 to about 800
0 Dalton, more preferably about 6000 to about 7000
Dalton. In the peptide in the present specification, the left end is the N-terminus (amino terminus) and the right end is the C-terminus (carboxyl terminus) according to the convention of peptide labeling. In the polypeptide of the present invention, the C-terminus usually has a carboxyl group (—COO).
H) or carboxylate (—COO ⁻ ),
C-terminal amide (-CONH ₂₎ or ester (-C
OOR). Examples of R in the ester include C _1-6 alkyl groups such as methyl, ethyl, n-propyl, isopropyl and n-butyl, C _3-8 cycloalkyl groups such as cyclopentyl and cyclohexyl, and C _3-8 such as phenyl and α-naphthyl. Phenyl-C such as _6-12 aryl group, benzyl, phenethyl, benzhydryl, etc.
_1-2 alkyl or α- such as α-naphthylmethyl
In addition to C _7-14 aralkyl groups such as naphthyl-C _1-2 alkyl and the like, pivaloyloxymethyl groups widely used as oral esters and the like can be mentioned. When the polypeptide of the present invention has a carboxyl group or a carboxylate other than at the C-terminus, those in which those groups are amidated or esterified are also included in the polypeptide of the present invention.
As the ester at this time, for example, the same ester as the above-mentioned C-terminal ester and the like are used.

As a salt of the polypeptide of the present invention, a salt with a physiologically acceptable base (for example, an alkali metal or the like) or an acid (organic acid or inorganic acid) is used, and especially physiologically acceptable. Acid addition salts are preferred. Such salts include, for example, salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid) or organic acids (eg, acetic acid,
For example, salts with formic acid, propionic acid, fumaric acid, maleic acid, succinic acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid) are used. The polypeptide of the present invention can be produced by a method for purifying a peptide from human or other warm-blooded animal tissues or cells, or can be produced according to the peptide synthesis method described later. The polypeptide of the present invention can also be produced by culturing a transformant containing the DNA encoding the polypeptide. DNA encoding the peptide can be obtained by a known cloning method [for example, Molecular Cloning (2nd ed .; JS
ambrook et al., Cold Spring Harbor Lab. Press, 198
9), etc.]. The cloning method includes (1) a DNA probe designed based on the amino acid sequence of the peptide or D
Using the NA primers, the cDNA encoding the peptide
A method for obtaining a transformant containing NA, or (2) a method for obtaining a transformant containing DNA encoding the peptide by PCR using a DNA primer designed based on the amino acid sequence of the peptide. . When the polypeptide of the present invention is produced from human or other warm-blooded animal tissues or cells, the human or other warm-blooded animal tissues or cells are homogenized, and then extracted with an acid or alcohol to obtain. The extract can be purified and isolated by combining chromatography such as salting out, dialysis, gel filtration, reverse phase chromatography, ion exchange chromatography and affinity chromatography.

As described above, the polypeptide of the present invention comprises
(1) According to a known peptide synthesis method, or (2)
It can be produced by cleaving a peptide containing the polypeptide 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 target peptide can be produced by condensing a partial peptide or amino acid capable of constituting the polypeptide of the present invention with the remaining portion and, when the product has a protecting group, removing the protecting group. Known condensation methods and elimination of protecting groups include, for example, the methods described in (1) to (4) below. M. Bodanszky and MA Ondetti, Peptide Synthesis, Interscience Publishe
rs, New York (1966); Schroeder and Luebke, The Peptid
e), Academic Press, New York (1965). After the reaction, the polypeptide of the present invention can be purified and isolated by a combination of ordinary purification methods such as solvent extraction, distillation, column chromatography, liquid chromatography, and recrystallization. When the peptide obtained by the above method is a free form, it can be converted to an appropriate salt by a known method, and when it is obtained by a salt, it can be converted to a free form by a known method. .

As the amide of the polypeptide of the present invention, a commercially available resin for peptide synthesis suitable for amide formation can be used. Examples of such a resin include chloromethyl resin, hydroxymethyl resin, benzhydrylamine resin, aminomethyl resin, 4-benzyloxybenzyl alcohol resin, 4-methylbenzhydrylamine resin, PAM resin, and 4-hydroxymethylmethylphenyl. Acetamidomethyl resin, polyacrylamide resin, 4
-(2 ', 4'-dimethoxyphenyl-hydroxymethyl) phenoxy resin, 4- (2', 4'-dimethoxyphenyl-Fmocaminoethyl) phenoxy resin and the like. Using such a resin, the amino acid appropriately protected α-amino group and side chain functional group, according to the sequence of the target peptide, according to various known condensation methods,
Condensed on resin. At the end of the reaction, the peptide is cleaved from the resin, and at the same time, various protecting groups are removed, and if necessary, an intramolecular disulfide bond formation reaction is carried out in a highly diluted solution to obtain a target peptide. For the condensation of the protected amino acids described above, various activating reagents that can be used for peptide synthesis can be used, and carbodiimides are particularly preferable. As carbodiimides, DCC,
N, N'-diisopropylcarbodiimide, N-ethyl-N'-3-dimethylaminoprolyl) carbodiimide and the like. Activation by these involves adding a protected amino acid directly to the resin along with a racemization inhibitor additive (eg, HOBt, HOOBt, etc.), or
Symmetric acid anhydride or HOBt ester or HOOB
The t-ester can be added to the resin after activation of the protected amino acid beforehand. 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 condensation reaction. For example, N, N-dimethylformamide, N, N-dimethylacetamide,
Acid amides such as N-methylpyrrolidone; halogenated hydrocarbons such as methylene chloride and chloroform; alcohols such as trifluoroethanol; sulfoxides such as dimethylsulfoxide; tertiary amines such as pyridine; dioxane; Ethers, 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 the peptide bond formation reaction, and is usually appropriately selected from a range of about -20 ° C to about 50 ° C. The activated amino acid derivative is usually used in about 1.5 to about 4-fold excess. As a result of the test using the ninhydrin reaction, when the condensation is insufficient, sufficient condensation can be performed by repeating the condensation reaction without removing the protecting group. When sufficient condensation cannot be obtained even by repeating the reaction, the unreacted amino acid can be acetylated using acetic anhydride or acetylimidazole so as not to affect the subsequent reaction. As the protecting group for the amino group of the starting amino acid,
For example, Z, Boc, tertiary pentyloxycarbonyl, isobornyloxycarbonyl, 4-methoxybenzyloxycarbonyl, Cl-Z, Br-Z, adamantyloxycarbonyl, trifluoroacetyl,
Examples include phthaloyl, formyl, 2-nitrophenylsulfenyl, diphenylphosphinothioyl, Fmoc and the like. Examples of the carboxyl-protecting group include, as R, the above-mentioned C _1-6 alkyl group, C _3-8 cycloalkyl group, C _7-14 aralkyl group, 2-adamantyl,
-Nitrobenzyl, 4-methoxybenzyl, 4-chlorobenzyl, phenacyl group and benzyloxycarbonyl hydrazide, tert-butoxycarbonyl hydrazide, trityl hydrazide and the like. The hydroxyl groups of serine and threonine can be protected, for example, by esterification or etherification. Suitable groups for this esterification include, for example, lower (C _1-6 ) alkanoyl groups such as acetyl group, aroyl groups such as benzoyl group, and groups derived from carbonic acid such as benzyloxycarbonyl group and ethoxycarbonyl group. . Examples of groups suitable for etherification include a benzyl group, a tetrahydropyranyl group, and a tertiary butyl group. Examples of the protecting group for the phenolic hydroxyl group of tyrosine include Bzl, Cl ₂ -Bzl, 2-nitrobenzyl, Br-Z, tertiary butyl and the like. The protecting group for imidazole of histidine includes To
s, 4-methoxy-2,3,6-trimethylbenzenesulfonyl, DNP, benzyloxymethyl, Bum, B
oc, Trt, Fmoc and the like. Examples of activated carboxyl groups of the raw materials include, for example, corresponding acid anhydrides, azides, active esters [alcohols (eg, pentachlorophenol, 2,4,5-trichlorophenol, 2,4-dinitrophenol, cyanophenol, Methyl alcohol, para-nitrophenol, HONB,
N-hydroxysuccinimide, N-hydroxyphthalimide, ester with HOBt)]. Examples of the activated amino group of the raw material include a corresponding phosphoric amide. Removal (elimination) of protecting group
Examples of the method include catalytic reduction in a hydrogen stream in the presence of a catalyst such as Pd-black or Pd-carbon, hydrogen anhydride, methanesulfonic acid, trifluoromethanesulfonic acid, trifluoroacetic acid, or a mixture thereof. Acid treatment, a base treatment with diisopropylethylamine, triethylamine, piperidine, piperazine and the like, and a reduction with sodium in liquid ammonia. The elimination reaction by the acid treatment is generally performed at a temperature of -20 ° C to 40 ° C. In the acid treatment, anisole, phenol, thioanisole, metacresol, paracresol, dimethylsulfide,
It is effective to add a cation scavenger such as 1,4-butanedithiol and 1,2-ethanedithiol. In addition, 2,4 used as an imidazole protecting group of histidine is used.
The dinitrophenyl group is removed by thiophenol treatment, and the formyl group used as the indole protecting group of tryptophan is 1,2-ethanedithiol as described above;
In addition to deprotection by an acid treatment in the presence of 1,4-butanedithiol or the like, it is also removed by an alkali treatment with dilute sodium hydroxide, dilute ammonia or the like.

The protection and protection of functional groups that should not be involved in the reaction of the raw materials, the elimination of the protective groups, the activation of the functional groups involved in the reaction, and the like can be appropriately selected from known groups or known means. . As another method for obtaining the amide form of the polypeptide of the present invention, first, after amidating the α-carboxyl group of the carboxyl terminal amino acid, extending the peptide chain to a desired chain length on the amino group side, A peptide (or amino acid) from which only the N-terminal α-amino group protecting group was removed and a peptide (or amino acid) from which only the C-terminal carboxyl group protecting group was removed, and these peptides were mixed in a solvent mixture as described above. Condensation in Details of the condensation reaction are the same as described above. After purifying the protected peptide obtained by the condensation, all the protecting groups are removed by the above-mentioned method to obtain a desired crude peptide. This crude peptide is purified by using various known purification means, and the main fraction is freeze-dried to obtain an amide of the desired peptide. To obtain an ester of the polypeptide of the present invention, the α-carboxyl group of the carboxy terminal amino acid is condensed with a desired alcohol to form an amino acid ester, and then an ester of the desired peptide is obtained in the same manner as the amide of the peptide be able to. As the polypeptide of the present invention, the above-mentioned SEQ ID NO: 17, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 34 or SEQ ID NO: 62 (preferably, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: : 34 or SEQ ID NO: 62), the same effect (eg, LH secretion regulating activity (effect), GALR1
Agonist activity, GALR2 agonist activity or GA
Any peptide may be used as long as it has LR3 agonist activity). These actions (activity) can be measured by a known method or a method analogous thereto,
For example, it can be measured by the methods described in Reference Example 1, Reference Example 17, and Examples 1 to 3 described below. Such peptides include, for example, SEQ ID NO: 17, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 34 or SEQ ID NO: 62 (preferably SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: : 34 or SEQ ID NO: 62) or a peptide having an amino acid sequence in which 1 to 5 amino acids have been deleted, added (inserted) or substituted from the amino acid sequence or a partial sequence thereof. The precursor of the polypeptide of the present invention is a peptide encoding the ligand peptide of the present invention as a partial sequence thereof (however, galanin having the amino acid sequence represented by SEQ ID NO: 7, 8, 9, or 10 or a precursor thereof) ) Can be used.

As a precursor of the polypeptide of the present invention, specifically, for example, a peptide (or polypeptide) containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 29, etc. Is raised. Here, the “substantially identical amino acid sequence” is 70 to 9 amino acid sequences represented by SEQ ID NO: 29.
9.9%, more preferably 80-99.9%, even more preferably 90-99.9%, and most preferably 95-9%.
An amino acid sequence having 9.9% homology; Further, as the “substantially identical amino acid sequence”,
(I) 1 to 7 amino acids, preferably 1 to 5 amino acids, more preferably 1 to 3 amino acids in the amino acid sequence represented by SEQ ID NO: 29 have been substituted with other amino acids An amino acid sequence, (ii) one or more, preferably one or less, in the amino acid sequence represented by SEQ ID NO: 29;
An amino acid sequence in which at least one and at most five amino acids have been deleted, more preferably at least one and at most three amino acids have been deleted; (iii) SEQ ID NO:
An amino acid sequence in which 1 to 7 amino acids are added to the amino acid sequence represented by 29, preferably 1 to 5 amino acids, more preferably 1 to 3 amino acids is added (or inserted). The position of "substitution, deletion, addition (or insertion)" is not particularly limited as long as it is other than a region encoding the ligand peptide of the present invention as a partial sequence thereof. Specific examples of the peptide (or polypeptide) containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 29 include, for example,
(1) a peptide (or polypeptide) containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 30, (2) the same or substantially the same as the amino acid sequence represented by SEQ ID NO: 37 (3) a peptide (or polypeptide) containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 38, or the like; Is raised. As the partial peptide of the present invention, a peptide individually containing each domain of the polypeptide of the present invention may be used, but a partial peptide containing a plurality of domains at the same time may be used. Also,
Since the above partial peptide may be used as an antigen for preparing an anti-ligand peptide antibody,
(I) N-terminal peptide of the polypeptide of the present invention, C
A partial peptide in which one to several (preferably one to three, more preferably one or two) other amino acid residues have been added (or inserted) to a partial peptide such as a terminal peptide or a central peptide; 1) one to several (preferably 1 to 3, more preferably 1 or 2) amino acid residues from partial peptides such as the N-terminal peptide, C-terminal peptide and central peptide of the polypeptide of the present invention; Deleted partial peptide, (iii) one to several (preferably one to three, more preferably one to three) constituent amino acids of the partial peptide such as the N-terminal peptide, C-terminal peptide, central peptide and the like of the polypeptide of the present invention. And 1 or 2) are substituted with other amino acid residues.

More specifically, as the above partial peptide, for example, (i) Al
a Pro Ala His Arg Gly Arg Gly Gly Cys-NH ₂ (C of the peptide having the amino acid sequence represented by SEQ ID NO: 44)
A terminal amide); a peptide consisting of the amino acid sequence represented by SEQ ID NO: 11; and (iii) a peptide consisting of the amino acid sequence represented by SEQ ID NO: 11.
A peptide consisting of the amino acid sequence represented by No. 12, (i.
v) a peptide consisting of the amino acid sequence represented by SEQ ID NO: 13, (v) a peptide consisting of the amino acid sequence represented by SEQ ID NO: 14, (vi) a peptide consisting of the amino acid sequence represented by SEQ ID NO: 15, (Vii) SEQ ID NO:
A peptide consisting of the amino acid sequence represented by 16, (vi
ii) a peptide consisting of the amino acid sequence represented by SEQ ID NO: 17; (ix) a peptide consisting of the amino acid sequence represented by SEQ ID NO: 35; (x) a peptide consisting of the amino acid sequence represented by SEQ ID NO: 36; And (xi) a peptide consisting of the amino acid sequence represented by SEQ ID NO: 43. The partial peptide in this specification also has an amide (—CONH ₂ ) or ester (—COO) at the C-terminus.
R). Here, examples of the ester group are the same as those of the peptide described above. When the partial peptide has a carboxyl group or a carboxylate at a position other than the C-terminal, those in which those groups are amidated or esterified are also included in the partial peptide of the present invention.
As the ester at this time, for example, the above-mentioned C-terminal ester and the like are used. The partial peptide of the polypeptide of the present invention itself has the activity of the polypeptide of the present invention (for example, LH secretion regulation activity (action), LHRH secretion regulation activity (action), activating action on galanin receptor, etc.). You may have. The polypeptide of the present invention or a partial peptide thereof may be a fusion protein with a protein whose function or property is well known. Specific examples of the fusion protein include, for example, Ala Pro Ala used in Reference Example 11 described later.
His Arg Gly Arg Gly Gly Cys-NH ₂ (C-terminal carboxyl group of the amino acid sequence represented by SEQ ID NO: 44 (-C
OOH) amidated (-CONH ₂ )) and a peptide comprising an amino acid sequence represented by
And a fusion protein with Limpet Hemocyanin (KLH).

As the salts of the partial peptides of the polypeptide of the present invention, the same salts as the above-mentioned salts of the peptides can be used. The partial peptide of the polypeptide of the present invention or its amide, ester or salt thereof can be produced according to the same synthetic method as in the case of the above-mentioned peptide, or by cleaving the polypeptide of the present invention with an appropriate peptidase. . The DNA encoding the polypeptide of the present invention includes (I) an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 35 (for example, SEQ ID NO: 13 or the like) and LH
A peptide having secretory regulatory activity (action), or (II)
Any DNA may be used as long as it contains DNA encoding a peptide or the like having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 36. Further, any DNA may be used as long as it contains the DNA encoding the polypeptide of the present invention specifically described above. In addition, any of genomic DNA, genomic DNA library, cDNA derived from the above-mentioned tissue / cell, cDNA library derived from the above-described tissue / cell, 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, RNA from the aforementioned tissue / cell
Reverse Transcriptas directly using the prepared fraction
It can also be amplified by e Polymerase Chain Reaction (hereinafter abbreviated as RT-PCR method). More specifically, (1) a peptide containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 34 or SEQ ID NO: 62 DNA containing DNA,
(2) DNA that hybridizes with the sequence defined in (1) under high stringent conditions, (3) hybridization with the sequence defined in (1) and (2) due to degeneracy of the genetic code However, DNAs encoding polypeptides having the same amino acid sequence are used.
Hybridization can be performed according to a known method or a method analogous thereto. The high stringency conditions include, for example, 42 ° C., 50% formamide, 4 × SSPE (1 × SSPE = 150 mM NaCl,
_{10mM NaH 2 PO 4 · H 2} O, 1mM EDTA pH7.4), 5 × Denhardt's solution and 0.1% SDS. A specific example of a DNA containing a DNA encoding a peptide having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 31 includes the base sequence represented by SEQ ID NO: 32 Specific examples of DNA containing DNA encoding a peptide containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 33 include:
And a DNA containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 34. Specific examples of DNA containing DNA include DNA containing the base sequence represented by SEQ ID NO: 40, and the like, and are the same as or substantially the same as the amino acid sequence represented by SEQ ID NO: 62. Specific examples of the DNA containing the DNA encoding the peptide containing the same amino acid sequence include a DN containing the base sequence represented by SEQ ID NO: 64.
And DNA containing A. Examples of the DNA encoding the precursor of the polypeptide of the present invention include the above-mentioned precursor of the polypeptide of the present invention, specifically, SEQ ID NO:
A peptide (polypeptide) containing the same or substantially the same amino acid sequence as the amino acid sequence represented by 29 (for example, the amino acid sequence represented by SEQ ID NO: 30, SEQ ID NO: 37 or SEQ ID NO: 38); Any DNA may be used as long as it contains a DNA encoding A specific example of a DNA containing a DNA encoding a peptide (polypeptide) having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 29 is represented by SEQ ID NO: 27 Specific examples of DNA containing DNA encoding a peptide containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 30 include a DNA containing a DNA containing a base sequence, and the like. As a DNA containing the base sequence represented by SEQ ID NO: 28
A specific example of DNA containing DNA encoding a peptide having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 37 is SEQ ID NO: 37. And DNA containing a DNA containing the nucleotide sequence represented by SEQ ID NO: 38. As a specific example of the contained DNA, a DNA containing the base sequence represented by SEQ ID NO: 42
Examples include DNA containing NA.

The DNA encoding the polypeptide of the present invention (hereinafter, the polypeptide of the present invention, the precursor of the polypeptide of the present invention, and these partial peptides may be simply referred to as the polypeptide of the present invention) is as follows: Can also be produced by the genetic engineering technique described above. The genetic engineering technique is described in, for example, Molecular Cloning (2nd ed .; J. Sambro
ok et al., Cold Spring Harbor Lab. Press, 1989). When a commercially available library or kit is used, the method may be performed according to the method described in the attached instruction manual. The DNA encoding the polypeptide of the present invention is selected by synthesizing a DNA fragment based on the amino acid sequence of the peptide or a part thereof and labeling the probe by hybridization with a genomic DNA or cDNA library or the like. Can be. D which completely encodes the polypeptide of the present invention
As a means for cloning NA, synthetic D having a base sequence encoding a partial DNA of the polypeptide of the present invention is used.
Using the NA primer, the target DNA is amplified from a genomic DNA or cDNA library or the like by a known PCR method, or the DNA incorporated into an appropriate vector is, for example, a partial or entire region of the polypeptide of the present invention. Can be selected by hybridization with a DNA fragment having the following or labeled with a synthetic DNA. The cloned DNA encoding the polypeptide of the present invention can be used as it is depending on the purpose, or may be used after digesting with a restriction enzyme or adding a linker, if desired. The DNA has ATG as a translation initiation codon at its 5 'end and 3'
The terminal may have TAA, TGA or TAG as a translation stop codon. These translation initiation codon and translation termination codon can also be added using a suitable synthetic DNA adapter. An expression vector for the polypeptide of the present invention can be obtained, for example, by (A) cutting out a DNA fragment of interest from DNA (eg, cDNA) containing DNA encoding the polypeptide of the present invention, and (B) expressing the DNA fragment appropriately. It can be produced by ligating downstream of a promoter in a vector. As the vector, a plasmid derived from E. coli (eg, pBR322, pB
R325, pUC12, pUC13) and plasmids derived from Bacillus subtilis (eg, pUB110, pTP5, pC19)
4), yeast-derived plasmids (eg, pSH19, pSH1
5), bacteriophages such as λ phage, and animal viruses such as retrovirus, vaccinia virus, and baculovirus are used.

The promoter used in the present invention may be any promoter as long as it is appropriate for the host used for gene expression. When the host used for the transformation is an animal cell, SV40-derived promoter, retrovirus promoter, metallothionein promoter, heat shock promoter, cytomegalovirus promoter, SRα promoter and the like can be used. When the host is a bacterium belonging to the genus Escherichia, a trp promoter, a T7 promoter, a lac promoter, a recA promoter, a λP _L promoter, an lpp promoter, and the like, and when the host is a bacterium belonging to the genus Bacillus, an SPO1 promoter, an SPO2 promoter, a penP promoter, and the like. When the host is yeast, PHO5 promoter, PGK promoter, G
An AP promoter, an ADH1 promoter, a GAL promoter and the like are preferred. When the host is an insect cell, a polyhedrin promoter, a P10 promoter and the like are preferable. In addition to those described 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 may be used as the expression vector. it can. As a selection marker, for example, dihydrofolate reductase (hereinafter, dhfr)
[May be 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, selection can be made even on a thymidine-free medium.
If necessary, a signal sequence suitable for the host is added to the N-terminal side of the peptide or its partial peptide.
When the host is a bacterium belonging to the genus Escherichia, a phoA signal sequence, an OmpA signal sequence, and the like, when the host is a bacterium belonging to the genus Bacillus, an α-amylase signal sequence;
When the host is yeast, the subtilisin signal sequence or the like is a mating factor α (MFα) signal sequence or an invertase signal sequence. When the host is an animal cell, for example, an insulin signal sequence or α- An interferon signal sequence, an antibody molecule and a signal sequence can be used. Using the vector containing the DNA encoding the peptide thus constructed, a transformant can be produced.

As the host, for example, Escherichia, Bacillus, yeast, insects or insect cells, animal cells and the like are used. Examples of the genus Escherichia include Escherichia coli K12.DH1.
[Processings of the National Academy of Sciences of the USA (Proc. Natl. Acad. Sci. USA), 60, 160
(1968)], JM103 [Nucleic Acids Research, Vol. 9, 309]
(1981)], JA221 [Journal of Molecular Biolo
gy), 120, 517 (1978)], HB101
[Journal of Molecular Biology, 4
1, 459 (1969)], C600 [Genetics, 39, 440 (1954)] and the like. Examples of Bacillus genus bacteria include Bacillus subtilis MI114 [Gene, 24, 255 (1983)], 207-21 [Journal of Biochemistry (Journal of Biochemistry).
Chemistry, Vol. 95, 87 (1984)]. Examples of yeast include Saccharomyces cerevisiae AH22 and AH22.
^{22R -, NA87-11A, DKD-5D} , 20B-
12 and the like are used. As insects, for example, silkworm larvae are used [Maeda et al., Nature (Natur
e), 315, 592 (1985)]. As the insect cell, for example, when the virus is AcNPV, a cell line derived from a larva of Spodoptera (Spodoptera frugiperda cell;
Sf cells), MG1 cells derived from the midgut of Trichoplusia ni, High Five ^™ cells derived from eggs of Trichoplusia ni, cells derived from Mamestra brassicae, or Estigmena ac
Cells derived from rea are used. Virus is BmNP
In the case of V, a silkworm-derived cell line (Bombyx mori N; B
mN cells). Examples of the Sf cells include Sf9 cells (ATCC CRL 1711) and Sf21 cells [Vaughn, JL et al., In vitro, 1
3, pp. 213-217 (1977)]. Examples of animal cells include monkey COS-7 cells, Vero cells, Chinese hamster cells CH
O, DHFR gene deficient Chinese hamster cell C
HO (CHO / dhfr ^- cell), mouse L cell, mouse 3T3 cell, mouse myeloma cell, human HEK29
3 cells, human FL cells, 293 cells, C127 cells, B
ALB3T3 cells, Sp-2 / O cells and the like are used.

In order to transform Escherichia bacteria, for example, Processing of the National Academy of Sciences of the USA (Proc. Natl. Acad. Sci. USA), Vol. 69, 21
10 (1972) and Gene, 17, 107
(1982). To transform Bacillus sp., For example, Molecular and General Genetics (Molecular
& General Genetics), 168, 111 (197
This is performed according to the method described in 9) and the like. To transform yeast, for example, the Procesings of the National Academy of Sciences of Science
The USA (Proc. Natl. Acad. Sci. USA),
75, 1929 (1978). In order to transform insect cells or insects, for example, Bio / Technology, 6
Vol., Pp. 47-55 (1988). Transformation of animal cells is described, for example, in Virology, 52, 456 (197).
This is performed according to the method described in 3). As a method for introducing an expression vector into cells, for example, a lipofection method [Felgner, PL et al.
The National Academy of Sciences
Proceedings of the Nationa
l Academy of Sciences of the United States of Amer
ica), 84, 7413 (1987)], calcium phosphate method [Graham, FL and van der Eb, AJ Virology, 52, 456-467 (1973)], electroporation method [Nuemann, E. et al.
EMBO J., Volume 1, 841-84
5 (1982)]. Thus, a transformant transformed with the expression vector containing the DNA encoding the polypeptide of the present invention is obtained. In addition, as a method for stably expressing the polypeptide of the present invention using animal cells, there is a method of selecting, by clonal selection, cells in which the expression vector introduced into the animal cells is integrated into the chromosome. Specifically, a transformant is selected using the above-mentioned selection marker as an index.
Furthermore, a stable animal cell line having high expression ability of the polypeptide of the present invention can be obtained by repeatedly performing clonal selection on the animal cells obtained using the selection marker. Further, when the dhfr gene is used as a selection marker, the MTX concentration is gradually increased, and culture is performed. By selecting a resistant strain, the DNA encoding the polypeptide of the present invention together with the dhfr gene is obtained.
Can be amplified intracellularly to obtain a more highly expressed animal cell line. The above transformant is cultured under conditions in which a DNA encoding the polypeptide of the present invention can be expressed,
By producing and accumulating the polypeptide of the present invention, the polypeptide of the present invention can be produced. When culturing a transformant whose host is a genus Escherichia or Bacillus, a liquid medium is suitable as a medium to be used for the culture. Nitrogen sources, inorganic substances, etc. are contained. Examples of carbon sources include glucose, dextrin, soluble starch, and sucrose. Examples of nitrogen sources include ammonium salts, nitrates, corn steep liquor, peptone, casein, meat extract, soybean meal, potato extract, and the like. Alternatively, examples of the organic substance and the inorganic substance include calcium chloride, sodium dihydrogen phosphate, and magnesium chloride. In addition, yeast, vitamins, growth promoting factors and the like may be added. The pH of the medium is about 5
8 is desirable. As a medium for culturing the genus Escherichia, for example, 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 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 may 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 minimum medium [Bostian, KL et al.
Prossings of the National Academy of Sciences of the USA (Pr
Natl. Acad. Sci. USA), 77, 4505.
(1980)] and 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 a transformant whose host is an insect cell, Grace's Insect M
edium (Grace, TCC, Nature, 195,788
(1962)) to which an inactivated additive such as 10% bovine serum is appropriately added. The pH of the medium is about 6.2
It is preferable to adjust to 6.4. Culture is usually about 27 ° C
For about 3 to 5 days, and aeration and stirring are added as necessary. When culturing a transformant in which the host is an animal cell, the medium may be, for example, a MEM medium containing about 5 to 20% fetal bovine serum [Science, 122, 50
1 (1952)], DMEM medium [Virology (Viro
logy), 8, 396 (1959)], RPMI 16
40 Medium [The Journal of the America Medical Association
n Medical Association) 199, 519 (196
7)] 199 medium [Proceeding of the Society for the Biological Med (Proceeding of the Society for the Biological Medicine)
icine), Vol. 73, 1 (1950)]. Preferably, the pH is between about 6 and 8. The cultivation is usually carried out at about 30 ° C. to 40 ° C. for about 15 to 60 hours, and if necessary, aeration or stirring is added. In particular, when CHO (dhfr ⁻ ) cells and the dhfr gene are used as selection markers,
D containing dialyzed fetal bovine serum containing almost no thymidine
Preferably, a MEM medium is used. The polypeptide of the present invention can be separated and purified from the above culture by, for example, the following method.

When the polypeptide of the present invention is extracted from cultured cells or cells, the cells or cells are collected by a known method after culture, and suspended in an appropriate buffer.
After disrupting cells or cells by ultrasonication, lysozyme and / or freeze-thawing, a method of obtaining a crude peptide extract by centrifugation or filtration may be used as appropriate. In a buffer solution, a protein denaturant such as urea or guanidine hydrochloride, or Triton X-100 (registered trademark; hereinafter, TM
May be abbreviated. ) May be included. When the peptide is secreted into the culture solution, after culturing, the supernatant is separated from the cells or cells by a known method, and the supernatant is collected. Purification of the polypeptide of the present invention contained in the thus obtained culture supernatant or extract can be carried out by appropriately combining known separation and purification methods. As these known separation and purification methods, methods utilizing solubility such as salting out and solvent precipitation, dialysis, ultrafiltration, gel filtration, and SDS-
Methods that mainly use differences in molecular weight such as polyacrylamide gel electrophoresis, methods that use differences in charge such as ion-exchange chromatography, methods that use specific affinity such as affinity chromatography, reverse-phase high-performance liquid chromatography A method using a difference in hydrophobicity such as chromatography, a method using a difference in isoelectric points such as isoelectric focusing or chromatofocusing are used. When the thus-obtained polypeptide of the present invention is obtained in a free form, it can be converted to a salt by a known method or a method analogous thereto. It can be converted to a free form or another salt by a method or a method analogous thereto. The polypeptide of the present invention produced by the transformant can be arbitrarily modified or 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 presence of the polypeptide of the present invention thus produced can be measured by an enzyme immunoassay using a specific antibody or the like.

The use of the LH secretion regulator containing the polypeptide of the present invention or the DNA encoding the polypeptide of the present invention will be described in detail below. The polypeptide of the present invention or the DNA encoding the polypeptide of the present invention has a specific action of increasing the blood LH concentration (a LH secretion promoting action), Was increased in Zucker fatty rats in which an abnormality was observed in the leptin receptor (Example 3). In addition, since galanin having a high homology to the polypeptide of the present invention does not show an LH secretion promoting action, the LH secretion promoting action is a specific action of the polypeptide of the present invention not found in galanin. In addition, from Example 3 to be described later, leptin-induced blood L
One end of the mechanism for increasing the H concentration is carried by the polypeptide of the present invention. Accordingly, since the peptide of the present invention or the DNA encoding the polypeptide of the present invention has an LH secretion promoting effect, various diseases related to LH secretion deficiency (specifically, infertility, irregular menstruation, dysmenorrhea, etc.) , Amenorrhea, rare menstruation, premenstrual syndrome, menopause, pituitary dysfunction or obesity, etc.). That is, the LH secretion regulator containing the polypeptide of the present invention or the DNA encoding the polypeptide of the present invention can be used as a LH secretion promoter as infertility, irregular menstruation,
It can be used as a preventive or therapeutic drug for dysmenorrhea, amenorrhea, rare menstruation, premenstrual syndrome, menopause, pituitary dysfunction or obesity. In addition, LH secretion regulators containing the polypeptide of the present invention or the DNA encoding the polypeptide of the present invention are particularly useful for various diseases caused by abnormal leptin receptors (specifically, obesity, infertility, etc.). ) Is considered to be effective as a prophylactic / therapeutic agent. On the other hand, since the polypeptide of the present invention has high affinity for its receptor protein, desensitization to LH secretion occurs when the dose of the polypeptide of the present invention or the DNA encoding the polypeptide of the present invention increases. As a result, it also has the effect of suppressing LH secretion. In this case, the LH secretion regulator containing the polypeptide of the present invention or the DNA encoding the polypeptide of the present invention is LH secretion inhibitor as LH secretion inhibitor.
Various diseases related to hypersecretion (specifically, prostate cancer,
Prostatic hyperplasia, precocious puberty or LH-producing pituitary tumor). Hereinafter, the use of the LHRH secretion regulator containing the polypeptide of the present invention or the DNA encoding the polypeptide of the present invention will be described in detail. The polypeptide of the present invention or the DNA encoding the polypeptide of the present invention has an LH-RH secretion stimulating action, and has dysmenorrhea, amenorrhea, rare menstruation, premenstrual syndrome, polycystic ovary syndrome and the like. It can be used as a prophylactic / therapeutic agent for a sex hormone-dependent disease, a prophylactic / therapeutic agent for a disease such as Alzheimer's disease and immunodeficiency, and a prophylactic / therapeutic agent for infertility. Furthermore, it can be used as a preventive / therapeutic agent for benign or malignant tumors which are sex hormone-independent but LH-RH sensitive.

The LH secretion regulator or LHRH secretion regulator containing the polypeptide of the present invention or the DNA encoding the polypeptide of the present invention can be produced and prepared according to a conventional method.
Can be used. 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, generally recognized pharmaceutical practice of treating a polypeptide of the invention or a DNA encoding a polypeptide of the invention with physiologically acceptable carriers, flavors, excipients, vehicles, preservatives, stabilizers, binders, and the like. By mixing in the unit dose form required. The amount of the active ingredient in these preparations is such that an appropriate dose in the specified range can be obtained. When the DNA of the present invention is used, the DNA can be administered alone or after inserting it into an appropriate vector such as a retrovirus vector, an adenovirus vector, an adenovirus associated virus vector, or the like. Tablets, capsules and the like can be mixed with additives such as gelatin, corn starch,
Binders such as tragacanth gum, acacia, excipients such as crystalline cellulose, leavening agents such as corn starch, gelatin, alginic acid, lubricants such as magnesium stearate, sucrose, lactose or saccharin Sweetening agents, 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 conventional methods, 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. 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 ( ^TM ), HCO-50) may be used in combination. 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.

Further, a buffer (for example, a phosphate buffer,
Sodium acetate buffer), soothing agents (eg, benzalkonium chloride, procaine hydrochloride, etc.), stabilizers (eg, human serum albumin, polyethylene glycol, etc.), preservatives (eg, benzyl alcohol, phenol, etc.), antioxidants And the like. The prepared injection is usually filled in a suitable ampoule. The preparations obtained in this way are safe and low toxic, and can be used, for example, in mammals (eg, humans, mice, rats, guinea pigs, rabbits, sheep, pigs, cows, cats, dogs, monkeys,
Etc.). The dosage of the polypeptide of the present invention or the DNA encoding the polypeptide may vary depending on the symptoms and the like. However, when the peptide is orally administered, generally, for an adult (assuming a body weight of 60 kg), the amount is about 0.5 per day. 1 to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg.
When the peptide is administered parenterally (for example, by intravenous injection), the single dose varies depending on the administration subject, target organ, symptoms, administration method and the like. For an adult (assuming a body weight of 60 kg), the dose is 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 above dose per 60 kg can be administered in terms of the weight of the animal.

Hereinafter, the receptor (galanin receptor) for the polypeptide of the present invention will be described in detail.
As used herein, the galanin receptor includes warm-blooded animals (eg, mammals (eg, humans, rabbits, sheep, goats, rats, mice, guinea pigs, cows, horses,
Pigs), birds (eg, chickens, pigeons, ducks, geese, quails), etc.)
Pancreas, brain, kidney, liver, gonad, thyroid, gall bladder, bone marrow, adrenal gland, skin, muscle, lung, digestive tract, blood vessels, heart, etc.)
Or a G protein-coupled receptor protein derived from a cell or the like, wherein GALR1 (Proc. Natl. Aca
d. Sci. USA, 90, pp3845-3849 (1993); J. Mol. Neu
rosci., 6, pp33-41 (1995); FEBS Lett., 411, pp225
-230 (1997)) and SEQ ID NO: 2 as GALR2 (described above), GALR3 (J. Biol. Che
m., 272, pp31949-31952 (1997)) as SEQ ID NO:
Any amino acid sequence containing the same or substantially the same amino acid sequence as the amino acid sequence represented by 3 may be used. That is, as the receptor protein, in addition to the protein containing the amino acid sequence represented by SEQ ID NO: 1, 2 or 3,
Or about 90 to 99.9 with the amino acid sequence represented by 3
% Amino acid sequence containing SEQ ID NO:
Proteins having substantially the same activity as the protein containing the amino acid sequence represented by 1, 2, or 3 are exemplified.

The activities exhibited by these proteins include:
For example, ligand binding activity (for the polypeptide of the present invention), signal transduction activity and the like can be mentioned. Substantially the same indicates that their activities are the same in nature. These activities can be measured by a known method or a method analogous thereto, for example, the methods described in Reference Examples 1, 17 and Examples 1 to 3 described below. Therefore, quantitative factors such as strength of ligand binding activity and signal transduction activity and molecular weight of receptor protein may be different. Furthermore, in the receptor protein, the N-terminal Met has a protecting group (for example, formyl group,
Protected with a C _1-6 alkanoyl group such as an acetyl group, etc., or those obtained by oxidizing pyroglutamine at the glutamine residue at the N-terminus. those protected by a C _1-6 alkanoyl group such group), or sugar chains are also conjugated proteins such as glycoproteins bound. As the salt of the receptor protein,
The same as the above-mentioned salts of the polypeptide of the present invention can be mentioned. The receptor protein or a salt thereof or a partial peptide thereof can be produced from a tissue or a cell of a warm-blooded animal by a known method for purifying a protein, or a transformant containing a DNA encoding the polypeptide of the present invention described above. It can also be produced by the same method as that for culturing the body. Further, it can be produced according to the above-mentioned peptide synthesis method. As the partial peptide of the receptor protein, for example, a site of the G protein-coupled receptor protein molecule which is exposed outside the cell membrane is used. That is, it is a peptide containing a portion analyzed as an extracellular region (hydrophilic site) in the hydrophobicity plot analysis of the G protein-coupled receptor protein. Further, a peptide partially containing a hydrophobic (Hydrophobic) 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. As the salt of the partial peptide of the receptor protein, those similar to the above-mentioned salts of the peptide are used.

The DNA encoding the galanin receptor protein includes a nucleotide sequence encoding a galanin receptor protein having an amino acid sequence identical or substantially identical to the amino acid sequence of SEQ ID NO: 1, 2 or 3. Anything that does is acceptable. They are also genomic DNA, genomic DN
Any of A library, tissue / cell-derived cDNA, tissue / cell-derived cDNA library, and synthetic DNA may be used. The vector used for the library may be any of bacteriophage, plasmid, cosmid, phagemid and the like. Alternatively, it can be directly amplified by a known RT-PCR method using an RNA fraction prepared from a tissue or a cell. SEQ ID NOs: 1, 2 and 3
Specific examples of the DNA encoding the galanin receptor having the amino acid sequence of SEQ ID NO:
DNAs having the base sequences represented by 4, 5, and 6 are used.

Further, the method of the following Examples 1 to 3 or a method analogous thereto (specifically, the blood LH concentration or the blood LHRH in the method of the following Examples 1 to 3 or the method analogous thereto) Changes in density change and Example 1 described later
3 or a method analogous thereto, a method of comparing a change in blood LH concentration or a change in blood LHRH concentration when the test compound or GALP and the test compound are administered, etc.) GALP agonists or inhibitors (antagonists, antibodies,
(Including antisense polynucleotides and the like). Since the GALP agonist obtained by such screening has the same LH secretion-promoting action as the polypeptide of the present invention, the LH secretion-promoting agent containing the agonist is infertile, irregular menstruation, dysmenorrhea, amenorrhea It is useful as a prophylactic / therapeutic agent for illness, rare menstruation, premenstrual syndrome, menopause, pituitary dysfunction or obesity. The method for producing and using the LH secretion promoter is the same as the method for producing and using the polypeptide of the present invention described above. Similarly, a GALP inhibitor obtained by such screening has an LH secretion inhibitory action, and therefore, an LH secretion inhibitor containing the inhibitor is useful for prostate cancer,
It is useful as a prophylactic / therapeutic agent for benign prostatic hyperplasia, precocious puberty or LH-producing pituitary tumor. The method for producing and using the LH secretion promoter is the same as the method for producing and using the polypeptide of the present invention described above. Since the GALP agonist obtained by such screening has the same LHRH secretion promoting action as the polypeptide of the present invention, the LHRH secretagogue containing the agonist is dysmenorrhea, amenorrhea, rare menstruation, It can be used as a prophylactic / therapeutic agent for sex hormone-dependent diseases such as premenstrual syndrome and polycystic ovary syndrome, a prophylactic / therapeutic agent for diseases such as Alzheimer's disease and immunodeficiency, and a prophylactic / therapeutic agent for infertility. it can. Furthermore, it can be used as a preventive / therapeutic agent for benign or malignant tumors which are sex hormone-independent but LH-RH sensitive. Similarly, an inhibitor of GALP obtained by such screening has an inhibitory effect on LHRH secretion, and therefore, an LHRH secretion inhibitor containing the inhibitor can be used for hormone-dependent diseases, particularly sex hormone-dependent cancers (eg, prostate cancer, Uterine cancer, breast cancer, pituitary tumor, etc.), benign prostatic hyperplasia, endometriosis, uterine fibroids, precocious puberty, and useful as a preventive or therapeutic agent for contraception. The compounds of the present invention, even when used alone, show excellent effects, but in combination with other pharmaceutical preparations and therapies,
The effect can be further enhanced. Examples of the concomitant drug and therapy include “bioactive peptide having LHRH secretion regulating action”, “appetite suppressant or bioactive peptide having appetite suppressing action” and the like (hereinafter abbreviated as concomitant drug). However, the present invention is not limited to this. Further, in addition to the combined use, the compound of the present invention and a compound having a preferable effect (specifically, various effects described below) may be prepared so as to be contained in the same preparation as a mixture. Further, another drug may be administered in combination with another drug, or simultaneously or after a certain period of time has elapsed after the first drug administration. Preferred examples of the physiologically active peptide having LHRH secretion regulating activity include LH-RH derivatives, which are hormone-dependent diseases, particularly sex hormone-dependent cancers (eg, prostate cancer, uterine cancer, breast cancer, pituitary tumor, etc.). ), Prostatic hyperplasia, endometriosis, uterine fibroids, precocious puberty, dysmenorrhea, amenorrhea, rare menstruation, premenstrual syndrome, multiple ovarian syndrome and other sex hormone dependent diseases and contraception (Or, in the case of utilizing the rebound effect after drug withdrawal, infertility), an effective LH-RH derivative or salt thereof. In addition, sex hormone-independent but LH-
Effective LH for RH-sensitive benign or malignant tumors
—RH derivatives or salts thereof are also included. Specific examples of the LH-RH derivative or a salt thereof include, for example, Treatment with GnRH analog: Treatment with GnRH an
alogs: Controversies and perspectives) [Parthenon Publishing Group, Inc. (The Parthenon P
ublishing Group Ltd.) 1996], Tokuheihei 3-50
3165, JP-A-3-101695 and 7-9
Peptides described in Nos. 7334 and 8-259460. Examples of LH-RH derivatives include LH-RH agonists and LH-RH antagonists. Examples of LH-RH antagonists include, for example, those represented by the general formula [I] X-D2Nal-D4ClPhe-D3Pal-Ser-AB-Leu-C -Pro-DAlaNH2 wherein X is N (4H2-furoyl) Gly or NAc, and A is NMeTy
r, Tyr, Aph (Atz), a residue selected from NMeAph (Atz),
B is DLys (Nic), DCit, DLys (AzaglyNic), DLys (AzaglyF
ur), DhArg (Et2), a residue selected from DAph (Atz) and DhCi, and C represents Lys (Nisp), Arg or hArg (Et2), respectively.) Can be As the LH-RH agonist, for example, the general formula [I
I] 5-oxo-Pro-His-Trp-Ser-Tyr-Y-Leu-Arg-Pro-Z wherein Y is selected from DLeu, DAla, DTrp, DSer (tBu), D2Nal and DHis (ImBzl) Z is NH-C2H5 or
Gly-NH2, respectively]] or a salt thereof, or the like. In particular, Y is DLeu,
A peptide wherein Z is NH-C2H5 (ie, 5-oxo-Pro-His-Trp
Peptide A represented by -Ser-Tyr-DLeu-Leu-Arg-Pro-NH-C2H5; leuprorelin) or a salt thereof (eg, acetate)
Is preferred. These peptides can be produced by the method described in the above-mentioned literature or gazette or a method analogous thereto.

The meanings of the abbreviations used in the above general formulas [I] and [II] are as follows. N (4H2-furoyl) Gly: N-tetrahydrofuroylglycine residue NAc: N-acetyl group D2Nal: D-3- (2-naphthyl) alanine residue D4ClPhe: D-3- (4-chloro) phenylalanine residue D3Pal: D-3- (3-pyridyl) alanine residue NMeTyr: N-methyltyrosine residue Aph (Atz): N- [5 '-(3'-amino-1'H-1', 2 ', 4 '-
Triazolyl)] phenylalanine residue NMeAph (Atz): N-methyl- [5 '-(3'-amino-1'H-
1 ', 2', 4'-triazolyl)] phenylalanine residue DLys (Nic): D- (eN-nicotinoyl) lysine residue Dcit: D-citrulline residue DLys (AzaglyNic): D- (azaglycylnicotinoyl) )
Lysine residue DLys (AzaglyFur): D- (azaglycylfuranyl) lysine residue DhArg (Et2): D- (N, N'-diethyl) homoarginine residue DAph (Atz): DN- [5 '-( 3'-amino-1'H-1 ', 2',
4'-triazolyl)] phenylalanine residue DhCi: D-homocitrulline residue Lys (Nisp): (eN-isopropyl) lysine residue hArg (Et2): (N, N'-diethyl) homoarginine residue

In the present specification, the appetite suppressant is a drug that suppresses appetite by acting directly or indirectly on the appetite center, and specific examples thereof include, for example, central appetite Inhibitors and bioactive peptide-related substances. Here, the central appetite suppressant is
It is a drug that suppresses appetite by acting on α-adrenergic receptor, β-adrenergic receptor, dopamine receptor or serotonin receptor. Preferred examples of the central anorectic include, for example, α-adrenergic receptor antagonists (eg, yohimbine), β-adrenergic receptor agonists (eg, mazindol, amphetamine, dextroamphetamine, phentermine,
Benzphetamine, methamphetamine, phendimetrazine, phenmetrazine, diethylpropion, sibutramine, phenylpropanolamine, clobenzolex, etc.), dopamine receptor agonists (eg, ER
-230, doprexin, etc.), serotonin receptor agonists (eg, dexfenfluramine, fenfluramine, etc.), 5-HT agonists (eg,
(+) Norfenfluramine, sertraline
ne), cimetidine, ergoset and the like.

Examples of the physiologically active peptide-related substance include a physiologically active peptide or an analog thereof, which acts directly or indirectly on the appetite center to suppress appetite, and an agonist or antagonist of the biologically active peptide. . Preferable examples of the physiologically active peptide-related substance include, for example, leptin and its analog, leptin receptor agonist, leptin resistance improving agent, neuropeptide Y (NPY / neuropeptide Y) antagonist (eg, NGD-95-1, SR -120819-A, PD-16017
0, 1229-U-91, etc.), cholecystokinin (CCK) agonists (eg, FPL-15849, GW-5823, GW-7178, GI-24857
3, AR-R-19021, etc.), glucagon-like peptide 1 (GL
P-1 / glucagon-like peptide 1) or its analog or its agonist (eg, AZM-134 etc.), galanin antagonist, glucagon agonist, melanin-concentrating hormone (MCH / melanin-
concentrating hormone antagonists, melanocortin agonists (especially melanocortin 4)
(Melacortin 4) receptor (MC4R) agonist, MC
4R / MC3R mixed agonist), enterostatin agonist, tripeptidyl peptidase II inhibitor (eg, UCL-1397), corticotropin releasing hormone or an analog thereof or an agonist thereof (eg, urocortin). Appetite suppressant is preferably a central appetite suppressant, more preferably a β-adrenergic receptor agonist,
Particularly preferred is mazindol.

In the present specification and drawings, when bases and amino acids are indicated by abbreviations, the IUPAC-IUB Commission
This is based on the abbreviation by on Biochemical Nomenclature or the abbreviation commonly used in this field, and examples thereof are described 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 I: inosine H: adenine, thymine or cytosine D: guanine, adenine or thymine B: guanine, thymine or cytosine RNA: ribonucleic acid mRNA: messenger ribonucleic acid dATP: deoxyadenosine triphosphate dTTP: deoxythymidine Triphosphate dGTP: deoxyguanosine triphosphate dCTP: deoxycytidine triphosphate ATP: adenosine triphosphate EDTA: ethylenediaminetetraacetic acid S S: sodium dodecyl sulfate EIA: enzyme immunoassay Gly or G: glycine Ala or A: alanine Val or V: valine Leu or L: leucine Ile or I: isoleucine Ser or S: serine Thr or T: threonine Cys or C: cysteine Or M: methionine Glu or E: glutamate Asp or D: aspartic acid Lys or K: lysine Arg or R: arginine His or H: histidine Phe or F: phenylalanine Tyr or Y: tyrosine Trp or W: tryptophan Pro or P: Asn or N: asparagine Gln or Q: glutamine pGlu: pyroglutamic acid Me: methyl group Et: ethyl group B u: butyl group Ph: phenyl group TC: thiazolidine-4 (R) -carboxamide group

The substituents, protecting groups and reagents frequently used in the present specification are represented by the following symbols. Tos: p-toluenesulfonyl HONB: N-hydroxy-5-norbornene-2,3
- dicarboximide Bzl: benzyl Cl ₂ -Bzl: di-chlorobenzyl Z: benzyloxycarbonyl Br-Z: 2-bromobenzyloxycarbonyl Cl-Z: 2-chloro benzyloxycarbonyl Boc: t-butyloxycarbonyl HOBt: 1 -Hydroxybenztriazole DCC: N, N'-dicyclohexylcarbodiimide TFA: trifluoroacetic acid Fmoc: N-9-fluorenylmethoxycarbonyl DNP: dinitrophenyl Bum: tert-butoxymethyl Trt: trityl PAM: phenylacetamidomethyl BHA: benz Hydrylamine Bom: benzyloxymethyl OcHex: cyclohexyl ester MeBzl: 4-methylbenzyl CHO: formyl NMP: N-methylpyrrolidone

The sequence numbers in the sequence listing in the present specification indicate the following sequences. [SEQ ID NO: 1] This shows the entire amino acid sequence of rat-type GALR1 (galanin receptor type 1). [SEQ ID NO: 2] This shows the entire amino acid sequence of rat GALR2 (galanin receptor type 2). [SEQ ID NO: 3] This shows the entire amino acid sequence of rat GALR3 (galanin receptor type 3). [SEQ ID NO: 4] This shows the nucleotide sequence of rat GALR1 (galanin receptor type 1) cDNA. [SEQ ID NO: 5] This shows the nucleotide sequence of rat GALR2 (galanin receptor type 2) cDNA. [SEQ ID NO: 6] This shows the base sequence of rat-type GALR3 (galanin receptor type 3) cDNA. [SEQ ID NO: 7] This shows the entire amino acid sequence (29 residues) of porcine galanin. [SEQ ID NO: 8] Pig-type galanin precursor prerogalani
The amino acid sequence of n (1-123) is shown. [SEQ ID NO: 9] Pig-type galanin precursor prerogalani
1 shows the amino acid sequence of n (24-61). [SEQ ID NO: 10] Pig-type galanin precursor preprogala
Fig. 4 shows the amino acid sequence of nin (37-61). [SEQ ID NO: 11] This shows the partial amino acid sequence (34 residues from N-terminal) of pig-type peptide of the present invention. [SEQ ID NO: 12] This shows the partial amino acid sequence (32 residues from N-terminal) of pig-type peptide of the present invention. [SEQ ID NO: 13] Partial amino acid sequence (N) of chymotrypsin digested fragment (CHY-1) of porcine-type peptide of the present invention
9 residues from the end). [SEQ ID NO: 14] Partial amino acid sequence of chymotrypsin digested fragment (CHY-4) of porcine peptide of the present invention (6)
Residue). [SEQ ID NO: 15] Partial amino acid sequence of chymotrypsin digested fragment (CHY-3) of pig-type peptide of the present invention (2)
7 residues). [SEQ ID NO: 16] Partial amino acid sequence (1) of chymotrypsin digested fragment (CHY-2) of pig-type peptide of the present invention
1 residue). [SEQ ID NO: 17] This shows the partial amino acid sequence (44 residues) of the pig-type peptide of the present invention. [SEQ ID NO: 18] Synthetic D used for screening of nucleotide sequence of cDNA encoding rat GALR2
NA (primer 1) is shown. [SEQ ID NO: 19] Synthetic D used for screening of the nucleotide sequence of cDNA encoding rat GALR2
NA (primer 2) is shown. [SEQ ID NO: 20] Degenerate PCR (Degenerate PCR)
d PCR) synthetic DNA (primer pGAL4-7
F) is shown. [SEQ ID NO: 21] Degenerate PCR (Degenerate PCR)
d PCR) synthetic DNA (primer pGAL9-3
F) is shown. [SEQ ID NO: 22] Degenerate PCR (Degenerate PCR)
d PCR) synthetic DNA (primer pGAL34-1
R). [SEQ ID NO: 23] This shows the base sequence of nested PCR product in pCR100-6. [SEQ ID NO: 24] This shows the base sequence of the nested PCR product in pCR100-7. [SEQ ID NO: 25] This shows the synthetic DNA (primer pGAL9-3F) used for preparing the hybridization probe. [SEQ ID NO: 26] This shows the synthetic DNA (primer pGAL34-8R) used for preparing the hybridization probe. [SEQ ID NO: 27] pGR2PL6 obtained in Reference Example 5
1 shows the nucleotide sequence of the cDNA in FIG. [SEQ ID NO: 28] pGR2PL3 obtained in Reference Example 5
1 shows the nucleotide sequence of the cDNA in FIG. [SEQ ID NO: 29] This shows the sequence of the precursor of the peptide of the present invention deduced from the nucleotide sequence of cDNA in pGR2PL6. [SEQ ID NO: 30] This shows the sequence of the precursor of the peptide of the present invention deduced from the nucleotide sequence of cDNA in pGR2PL3. [SEQ ID NO: 31] pGR2PL6 and pGR2PL
3 shows the sequence of the mature protein (porcine type) of the peptide of the present invention deduced from the nucleotide sequence of the cDNA in 3. [SEQ ID NO: 32] This shows the cDNA sequence (porcine type) encoding the mature protein of the peptide of the present invention. [SEQ ID NO: 33] This shows the sequence of the mature protein (rat type) of the peptide of the present invention. [SEQ ID NO: 34] This shows the sequence of the mature protein (human type) of the peptide of the present invention. [SEQ ID NO: 35] This shows the partial amino acid sequence (9 residues from N-terminus) of mature protein (common to rat type and human type) of peptide of the present invention. [SEQ ID NO: 36] This shows the partial amino acid sequence (21 residues from the N-terminus) of mature protein of the peptide of the present invention (common to rat type and human type). [SEQ ID NO: 37] This shows the amino acid sequence of the precursor protein of the mature protein (rat type) of the peptide of the present invention. [SEQ ID NO: 38] This shows the amino acid sequence of the precursor protein of the mature protein (human type) of the peptide of the present invention. [SEQ ID NO: 39] This shows the cDNA sequence encoding the mature protein (rat type) of the peptide of the present invention. [SEQ ID NO: 40] This shows the cDNA sequence encoding the mature protein (human type) of the peptide of the present invention. [SEQ ID NO: 41] cDNA encoding precursor protein of mature protein (rat type) of peptide of the present invention
Shows the sequence. [SEQ ID NO: 42] This shows the cDNA sequence encoding the precursor protein of the mature protein (human type) of the peptide of the present invention. [SEQ ID NO: 43] Partial amino acid sequence of mature protein (porcine type) of peptide of the present invention (30 residues from N-terminus)
Is shown. [SEQ ID NO: 44] This shows the amino acid sequence of the peptide used as an immunogen in Reference Example 9. [SEQ ID NO: 45] Plasmid used in Reference Example 18
Fig. 4 shows the DNA sequence of the EcoRI / BglII digested fragment of pGR2PL6. [SEQ ID NO: 46] Primer used in Reference Example 18
1 shows the DNA sequence of pGAL1-1F. [SEQ ID NO: 47] Primer used in Reference Example 18
Fig. 3 shows the DNA sequence of pGAL88-1R. [SEQ ID NO: 48] Primer used in Reference Example 19
1 shows the DNA sequence of F / R120. [SEQ ID NO: 49] Primer used in Reference Example 19
1 shows the DNA sequence of R / R120. [SEQ ID NO: 50] This shows the cDNA sequence of the result of PCR in Reference Example 19. [SEQ ID NO: 51] Primer used in Reference Example 19
1 shows the DNA sequence of 1F / H120. [SEQ ID NO: 52] Primer used in Reference Example 19
1 shows the DNA sequence of 1R / H120. [SEQ ID NO: 53] Primer used in Reference Example 19
1 shows the DNA sequence of 1F / H470. [SEQ ID NO: 54] Primer used in Reference Example 19
1 shows the DNA sequence of 1R / H470. [SEQ ID NO: 55] This shows the DNA sequence of primer 1 used in Reference Example 20. [SEQ ID NO: 56] This shows the cDNA sequence of Primer 2 used in Reference Example 20. [SEQ ID NO: 57] This shows the DNA sequence of primer 3 used in Reference Example 20. [SEQ ID NO: 58] This shows the DNA sequence of Primer 4 used in Reference Example 20. [SEQ ID NO: 59] Synthetic DNA used in Reference Example 21
Shows the sequence. [SEQ ID NO: 60] Synthetic DNA used in Reference Example 21
Shows the sequence. [SEQ ID NO: 61] This shows the amino acid sequence of rat galanin (rat galanin is a peptide having the amino acid sequence represented by SEQ ID NO: 61, in which the C-terminal is amidated). [SEQ ID NO: 62] This shows the amino acid sequence of mature mouse GALR2 ligand of the present invention. [SEQ ID NO: 63] This shows the amino acid sequence of the mouse GALR2 ligand precursor of the present invention. [SEQ ID NO: 64] This shows the base sequence of cDNA of mouse GALR2 ligand of the present invention. [SEQ ID NO: 65] Mouse GALR2 ligand c
1 shows the nucleotide sequence of a primer used for PCR for cloning DNA. [SEQ ID NO: 66] Mouse GALR2 ligand c
1 shows the nucleotide sequence of a primer used for PCR for cloning DNA. [SEQ ID NO: 67] This shows the base sequence of cDNA cloned using primers of SEQ ID NOs: 4 and 5. [SEQ ID NO: 68] Mouse GALR2 ligand c
The base sequence of the primer used for 5'RACE for cloning DNA is shown. [SEQ ID NO: 69] c of mouse GALR2 ligand
This shows the base sequence of the primer used for 3′RACE for cloning DNA. [SEQ ID NO: 70] Mouse type GALR2 ligand c
1 shows the nucleotide sequence of a primer used for PCR for cloning DNA. [SEQ ID NO: 71] c of mouse GALR2 ligand
1 shows the nucleotide sequence of a primer used for PCR for cloning DNA. [SEQ ID NO: 72] This shows the base sequence of cDNA of mouse GALR2 ligand precursor of the present invention.

The transformant Esch obtained in Reference Example 5 described later
erichia coli TOP10 / pGR2PL6 is available from 2-17-17, Jusanhoncho, Yodogawa-ku, Osaka-shi, Osaka (postal code 532-868).
6) with the Fermentation Research Institute (IFO) as the deposit number IFO16201 from August 21, 1998, and 1-1-1, Higashi 1-chome, Tsukuba, Ibaraki Prefecture, Chuo No. 6 (zip code 3
05-8566) National Institute of Advanced Industrial Science and Technology
Patent Organism Depositary Center (formerly Ministry of International Trade and Industry, National Institute of Advanced Industrial Science and Technology (NIBH)) September 4, 1998
It has been deposited from the date as deposit number FERM BP-6486. The transformant Esch obtained in Reference Example 19 described later
erichia coli TOP10 / pGR2HL14 is available from 2-17-17, Jusanhoncho, Yodogawa-ku, Osaka-shi, Osaka (postal code 532-868).
6) to the Fermentation Research Institute (IFO) as a deposit number IFO16256 from February 5, 1999, and 1-1-1, Higashi 1-chome, Tsukuba, Ibaraki Prefecture, Chuo No. 6 (zip code 30
5-8566) at the National Institute of Advanced Industrial Science and Technology (AIST) at the Patent Organism Depositary Center (formerly Ministry of International Trade and Industry, National Institute of Advanced Industrial Science and Technology (NIBH)), February 22, 1999.
It has been deposited from the date of deposit number FERM BP-6657. The transformant Esch obtained in Reference Example 18 described later
erichia coli TOP10 / pGR2RL4 is available at 2-17-85, Jusanhoncho, Yodogawa-ku, Osaka-shi, Osaka (postal code 532-868).
6) with the Fermentation Research Institute (IFO) as a deposit number IFO16257 from February 5, 1999, and at 1-1 1-1 Higashi, Tsukuba City, Ibaraki Prefecture, Chuo No. 6 (zip code 30
5-8566) at the National Institute of Advanced Industrial Science and Technology (AIST) at the Patent Organism Depositary Center (formerly Ministry of International Trade and Industry, National Institute of Advanced Industrial Science and Technology (NIBH)), February 22, 1999.
It has been deposited from the date of deposit number FERM BP-6658. The transformant Esch obtained in Reference Example 21 described later
erichia coli MM294 (DE3) / pTFCGAL is available from 17-1785 Jusanhoncho, Yodogawa-ku, Osaka-shi, Osaka (postal code 532-8).
686) to the Fermentation Research Institute (IFO) as a deposit number IFO 16260 from February 26, 1999, and an independent administrative corporation of Chuo No. 6 (zip code 305-8566), 1-1 1-1 Higashi, Tsukuba, Ibaraki Prefecture It has been deposited with the National Institute of Advanced Industrial Science and Technology at the Patent Organism Depositary (formerly Ministry of International Trade and Industry, National Institute of Advanced Industrial Science and Technology (NIBH)) as a deposit number FERM BP-6678 from March 10, 1999. The transformant Escherichia coli MM294 (DE3) / pTB used in Reference Example 21 described later
960-11 is 2-1-1 Jusanhoncho, Yodogawa-ku, Osaka-shi, Osaka
No. 7-85 (Zip code 532-8686) as a deposit number IFO 16100 from June 25, 1997 at the Fermentation Research Institute (IFO), as well as 1-1-1, Higashi 1-1, Tsukuba City, Ibaraki Pref. No. 305-856
6) 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)) under deposit number FERM BP-6388 from June 15, 1998. ing.
The transformant Escherichia used in Reference Example 21 described later
coli MM294 (DE3) / pTCIId23-MPI
F1 is 2-17-1785 Jusanhoncho, Yodogawa-ku, Osaka-shi, Osaka
No. (Zip code 532-8686) to the Fermentation Research Institute (IFO) from October 27, 1998
O16212, and also the National Institute of Advanced Industrial Science and Technology (AIST), Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology, 1-1 1-1 Higashi, Tsukuba City, Ibaraki Prefecture (formerly Ministry of International Trade and Industry) Research Institute (NIBH), deposited on Nov. 24, 1998 as accession number FERM BP-6582. GR2-1N used in Reference Example 13 to be described later is 2-17-85, Jusanhoncho, Yodogawa-ku, Osaka-shi, Osaka (postal code 532).
-8686) to the Institute for Fermentation (IFO) 199
From March 11, 2009, accession number IFO 50515 and 1-1-1, Higashi 1-chome, Chuo No. 6 Tsukuba, Ibaraki Prefecture
19th National Institute of Advanced Industrial Science and Technology (Postal Code 305-8566) Patent Organism Depositary Center (formerly National Institute of Advanced Industrial Science and Technology (NIBH))
Deposit No. FERM BP-668 from March 17, 1999
Deposited as No. 2. The transformant Escherichia coli TOP10 / pGR2ML1 obtained in Reference Example 25 described below was obtained from Fermentation Research Institute (IFO), 2-17-85, Jusanhoncho, Yodogawa-ku, Osaka-shi, Osaka (zip code 532-8686). From February 2, 2000 as deposit number IFO16361, and 1-1 1-1 Higashi 1-chome, Tsukuba, Ibaraki
(Postal code 305-8566) at the National Institute of Advanced Industrial Science and Technology, Patent Organism Depositary Center (formerly Ministry of International Trade and Industry, National Institute of Advanced Industrial Science and Technology, NIBH)
Deposit No. FERM BP-709 from March 16, 2000
Deposited as No. 2.

[0036]

The present invention will be described in more detail with reference to the following Reference Examples and Examples, which do not limit the scope of the present invention.

Reference Example 1 Galanin receptor (GAL)
R1, GALR2) Activation detection (1-1) Rat
Type galanin receptor (GALR1, GALR2)
Construction of current cells To obtain rat GALR2 gene, rat hypothalamus
2 μl of cDNA (CLONTECH) and 10 μM of primer
-1 (5'-GTCGACATGAATGGCTCCGGCAGCCAG-3 ', SEQ ID NO:
No .: 18) and primer 2 (5'-ACTAGTTTAACAAGCCG
GATCCAGGGTTCTAC-3 ′, SEQ ID NO: 19) was added in an amount of 1 μl,
0x concentrated buffer (with Klen Taq Polymerase manufactured by CLONTECH)
5 μl, 10 mM deoxynucleotide
1 μl of Domixture (Invitrogen), Klen Taq D
1 μl of NA polymerase (CLONTECH), distilled water for injection
(Otsuka Pharmaceutical Co., Ltd.) and mix PCR reaction solution.
Made. The PCR reaction was performed using the Gene Amp PCR System 9700 (PE
RKIN ELMER) at 95 ° C for 5 minutes.
(95 ° C, 30 seconds; 76 ° C, 15 seconds; 72 ° C,
2 minutes) for 3 cycles (95 ° C, 30 seconds; 72 ° C, 1
5 seconds; 72 ° C, 2 minutes) for 3 cycles (95 ° C, 30 minutes)
Second; 68 ° C., 15 seconds; 72 ° C., 2 minutes) for 3 cycles,
(95 ° C, 30 seconds; 64 ° C, 15 seconds; 72 ° C, 2 minutes)
3 cycles, (95 ° C., 30 seconds; 60 ° C., 15 seconds; 7
3 cycles of (2 ° C., 2 minutes), (95 ° C., 30 seconds; 56)
° C, 15 seconds; 72 ° C, 2 minutes) as 20 cycles.
The reaction was performed. 10 μl of the solution after the PCR reaction was added to 1
% Low melting point agarose gel (Sea Plaque GTG: FTN)
Perform the electrophoresis with PCR and convert the PCR reaction product to ethidium bromide.
It was confirmed by id staining. Obtained as the PCR reaction product
DNA band around 1.2 kbp was
From the agarose gel. That is, agarose
Transfer the gel fragment to a 0.5 ml sampling tube 7
After heating to 0 ° C and melting, equilibrating to 40 ° C, agarose
Beta agarase (Nipponji
0.5 μl was added and reacted for 60 minutes. The reaction solution
The desired DNA fragment by the ethanol precipitation method known from
Collect the PCR-SCRIPT (STRATAGENE) plasmid vector
Ligation reaction was performed. The reaction solution is
etent High (JM109: TOYOBO) and transformation
No, LB agar containing 100 μg / ml ampicillin
The cells were cultured overnight in a medium (Wako Pure Chemical Industries, Ltd.). Colony on the medium
-Insertion of target DNA fragment by colony-PCR method
It was confirmed. 100 colonies with the inserted DNA fragment
LB medium containing μg / ml ampicillin (Wako Pure Chemical Industries, Ltd.
) Overnight, and use the Plasmid Mini Kit
(QIAGEN) using a plasmid having an inserted DNA fragment.
Rasmid was recovered. The reaction for nucleotide sequence analysis is Dy
e primer cycle sequencing ready reaction (ABI)
Nucleotide sequencing using a fluorescent automatic sequencer
By column analysis, the inserted DNA fragment in the plasmid was 11
DNA encoding GALR2 consisting of 19 bp
It was confirmed. Reference of JP-A-7-304797
The expression vector pAKK for animal cells was obtained by the method described in Example 4.
O-1.11 was prepared. According to known methods, the plasmid
DNA fragments encoding GALR2 were recovered from the vector
-Linked to pAKKO-1.11, introduced into E. coli DH5α and transformed
A transformant was obtained. This transformant is cultured to obtain GALR2.
A plasmid containing the encoding DNA was prepared. The plastic
Use Sumid with CellPhect Transfection kit (Pharmacia)
CHO cells by the following procedure using
GALR2 expressing cells were obtained. First, 240 μl of distilled water
9.6 mg of plasmid DNA dissolved in
r A (attached to CellPhect Transfection Kit) 240μl
Is added, stirred, and allowed to stand for 10 minutes. Then, Buffer B (CellPh
ect Transfection Kit)
Stirring to form liposomes containing the DNA.
Was. 4 × 10^FiveCHO / dhfr^-Cells (ATCC
) In a 60 mm petri dish and 10% fetal bovine
Ham's F-12 medium containing serum (BIO WHITTAKER) (Nissui
Pharmaceutical Co., Ltd.) Cultivation for 2 days at 37 ° C and 5% CO2
After feeding, 480 μl of the liposome was added to the cells of a petri dish.
Dropped on top. This is at 37 ° C in 5% carbon dioxide.
After culturing for 6 hours, the cells were cultured in a serum-free Ham's F-12 medium for 2 hours.
The cells are washed once and 15% glycerol is placed on the cells in a Petri dish.
3 ml was added and the mixture was treated for 2 minutes. Then, again,
Washes twice with Ham's F-12 medium containing no
37 ° C, 5% charcoal in Ham's F-12 medium containing fetal bovine serum
The cells were cultured in acid gas for 15 hours. Treating the cells with trypsin
And collected from a Petri dish, and 1.25 × 10
^FourSeed each in a 6-well plate and dialyzed 10% bovine
Dulbecco'smodi containing fetal serum (JRH BIOSCIENCES)
fied Eagle medium (DMEM) medium (Nissui Pharmaceutical Co., Ltd.)
Incubation at 37 ° C in 5% CO2
Was. The transformed CHO cells into which the plasmid has been introduced are
Growing in the ground, but the non-transfected cells will gradually die
1 day and 2 days after the start of culture
Dead cells were removed. Traits that grew 8 days after the start of culture
Twenty colonies of transformed CHO cells (20 CHO cells)
Cell clone). Selected cells (20 species)
CHO cell clone) was collected and the following Reference Example (1-
A membrane fraction was prepared by the method described in 2). For membrane fraction
pig ¹²⁵Binding amount of I-galanin (New England Nuclear)
By a known method (for example, as described in Examples of EP-0711830A).
Cell line with high expression of GALR2
Was selected for use in subsequent experiments.

The cDNA encoding GALR1 was obtained in the same manner as the above-described method for obtaining the cDNA encoding GALR2. Using primers 3 and 4 rats, brain cD
Rat GALR1 cDNA consisting of 1486 bp was obtained from the NA library (CLONTECH) by PCR, inserted into pUC119 (Takara Shuzo Co., Ltd.), and named plasmid pRGR2. A cDNA fragment obtained by double digestion of this plasmid with restriction enzymes EcoRI and PstI was used as an expression plasmid vector pcDNA I for animal cells (Invitrogen
Was incorporated into the double-digested portion with restriction enzymes EcoRI and NsiI, and named plasmid pRGRPC. A cDNA fragment obtained by double digestion of this plasmid pRGRPC with restriction enzymes HindIII and XbaI was incorporated into an animal cell expression plasmid vector pRc / CMV (Invitrogen) double-digested with HindIII and XbaI, and rat GALR1 c
The DNA expression plasmid pRGR1 was obtained. CellPhect Transfe
Ham's F12 medium (Nissui Pharmaceutical Co., Ltd.) by the well-known calcium phosphate method using ction Kit (Pharmacia)
The rat GALR1 cDNA expression plasmid pRGR1 was introduced into CHO-K1 cells (obtained from ATCC) cultured in
Clones were isolated using stainless steel cylinders. A part of the cells of the 24 clones isolated in this manner were seeded on a 6-well plate, and cultured to saturation.
0 pM pig ¹²⁵ I-galanin (New England Nuclear
(For example, the method described in the example of EP-0711830A), and the expression level of rat galanin receptor in 24 clones was examined. As a result, no. 3 cells had the highest binding activity to porcine ¹²⁵ I-galanin,
It was considered that the expression level of GALR1 was the highest. Therefore,
No. was determined by the limiting dilution method. Three cells were seeded at a rate of one per two wells in a 96-well microplate and grown from one cell. Twelve clone cells were isolated from the singulated cells, and a similar binding experiment was performed again using some of the cells. As a result, no. 3-10 cloned cells have the highest binding activity with ¹²⁵ I-butagalanin, and GA
Since the expression level of LR1 was considered to be the highest, this clone was used as a GALR1-expressing cell.

(1-2) Galanin receptor (GAL)
Preparation of R1, GALR2) -expressing cell membrane fraction The GALR2-expressing cells obtained in (1-1) above were subconfluent (80 to 100%) in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, glutamine, penicillin, and streptomycin. (90% confluence). After culturing the cells, a phosphate buffer containing 2.7 mM EDTA [2.7 mM EDTA / Phosphate-buffer sali
ne (138 mM NaCl, 2.7 mM KCl, 8 mM Na ₂ HPO ₄ , 1 mM KH ₂ P
O ₄ )] and released from the incubator by suspending,
The cells were collected by centrifugation. The cells were treated with a protease inhibitor mixture (final concentrations 0.5 mM phenylmethylsulfonyl fluoride, 20 μg / ml leupeptin, 4 μg / ml E, respectively).
10m containing -64, 10 μg / ml pepstatin)
M sodium bicarbonate, 5 mM EDTA (pH 7.
3) Homogenized in a buffer using a Polytron homogenizer. Transfer the homogenate to a high-speed centrifuge (CR26
H, using RR24A rotor: Hitachi, Ltd.)
After centrifugation at 10 rpm for 10 minutes, the obtained supernatant was collected using an ultracentrifuge (SCP70H, RP42 type rotor: Hitachi, Ltd.).
Centrifugation was performed at 000 rpm for 1 hour to obtain a precipitate. This precipitate was re-produced with a protease inhibitor mixture (0.5 mM phenylmethylsulfonyl fluoride, 20 μg / ml leupeptin, 4 μg / ml E-
10 mM containing 64, 10 μg / ml pepstatin)
Sodium bicarbonate, 5 mM EDTA (pH 7.3)
The suspension was suspended in a buffer to prepare a membrane fraction for detection of galanin receptor activating action (stored at -70 ° C).

(1-3) Detection of Galanin Receptor Activating Action by [ ³⁵ S] GTPγS Binding Test
ml (for the GALR1 membrane fraction) or 32 μg / ml
(In the case of GALR2 membrane fraction), 1 μM guanosine-5′-diphosphate (GDP), 0.1% bovine serum albumin (BSA), 5 mM MgCl ₂ , 150 mM
50 mM Tris buffer (pH 7.0) containing mM NaCl.
4) and dispensed 0.2 ml each into small polypropylene test tubes (Falcon 2053). 5 in the dispensed membrane fraction
0 nM [ ³⁵ S] GTPγS (New England Nuclear)
μl and porcine galanin as agonist (100 μl
M, 10 μM, 1 μM, 100 nM, 10 nM, 1 n
2 μl (at each concentration of M, 100 pM, or 10 pM) was added and reacted at 25 ° C. for 60 minutes. In the reaction solution:
5 ml of 0.05% 3-[(3-cholamidopropyl) dimethylmonone] propanesulfonic acid (CHA
PS), 0.1% BSA, 5 mM MgCl ₂ , 1 mM
50 mM Tris buffer containing EDTA (pH 7.4)
Was added, and the mixture was filtered through GF / F glass fiber filter paper (Whatman). After the filter paper was washed with 1.5 ml of the same buffer and dried, the radioactivity was measured by a liquid scintillation counter. As a result, the galanin concentration
An increase in the amount of [ ³⁵ S] GTPγS binding was observed (FIG. 1).
It was revealed that the galanin receptor agonist activity can be measured by this method. Rat Galanin EC
_{The 50} value (concentration that gives half the maximum binding amount) is
It was about 3 nM for ALR1 and about 10 nM for GALR2.

Reference Example 2 Screening of Compound Activating GALR2 (Galanin Receptor Type 2) (2-1) Preparation of Swine Hypothalamic Extract The swine hypothalamus was purchased from Tokyo Shibaura Organ Co., Ltd. for 3 hours The following operation was performed within the above to prepare an extract. First, 35 pig hypothalamus (approximately 500 g) were finely chopped using a kitchen knife and contained 3000 m containing 1250 ml of boiling distilled water.
Put into a beaker and boiled for 10 minutes. After boiling the hypothalamus with a beaker in a water bath and then in an ice bath, bring the temperature to about 4 ° C.
Down to The hypothalamus was homogenized with distilled water used for boiling for 10 minutes using a Polytron homogenizer. 90 ml of acetic acid was added dropwise to the obtained homogenate so that the final concentration became 1 M, and the mixture was stirred for 1 hour. Transfer the homogenate to a high-speed centrifuge (CR26H, RR10A rotor:
Using Hitachi, Ltd.), the mixture was centrifuged at 10,000 rpm for 30 minutes to obtain a supernatant (1). On the other hand, the precipitate obtained after centrifugation was added again with 2000 ml of 1M acetic acid to obtain a polytron.
Homogenized using a homogenizer for 10 minutes. The homogenate was stirred overnight (about 16 hours) using a stirring blade, and then homogenized using a Polytron homogenizer for 10 minutes to obtain a supernatant (2). The supernatant (1) and the supernatant (2) are mixed, and twice the volume of acetone is added thereto.
And then use a high-speed centrifuge (CR26H, RR10A rotor: Hitachi, Ltd.) at 10,000 rpm.
and centrifuged for 15 minutes to obtain a supernatant. The obtained supernatant was applied to a rotary evaporator to remove acetone, and finally concentrated to 4000 ml. This concentrated solution was centrifuged at 35,000 rpm for 1 hour using an ultracentrifuge (SCP70H, Hitachi RPZ35T type rotor: Hitachi, Ltd.) to obtain a clear supernatant. The obtained supernatant was added to 500 ml every 1000 ml.
The mixture was mixed with 100 ml of diethyl ether, mixed vigorously in a separating funnel, and an aqueous phase was obtained after two-phase separation. The obtained aqueous phase was concentrated to 1000 ml using a rotary evaporator to obtain a final extract.

(2-2) Purification of porcine hypothalamic extract by octadodecyl reverse phase chromatography Silica gel ODS-AM 120-S50 having octadodecyl groups fixed
(YMC) was swelled with methanol, and then packed in a 5 cm diameter glass column so that the volume became 130 ml.
Equilibrated with acetic acid. The extract prepared in (2-1) (500 g under the thalamus) was applied to the column at a flow rate of 400 ml / h. Subsequently, about 500 ml of 1M
Acetic acid, then about 500 ml of 20% acetonitrile /
The gel was washed by flowing 0.1% trifluoroacetic acid at a flow rate of 400 ml / h. Finally, this column contains approximately 500
ml of 50% acetonitrile / 0.1% trifluoroacetic acid was flowed at a flow rate of 400 ml / h to elute the crude peptide component of interest. The obtained eluate was concentrated using an evaporator and then freeze-dried (12EL; VirTis
Lyophilized.

(2-3) TSKgel O of porcine hypothalamus extract
DS80 ^TM reverse-phase high performance liquid chromatography with purified TSKgel ODS80 ^TM reversed phase high performance liquid chromatography column (Tosoh Corporation, 22.5 mm × 30 cm) and 4
Solution A (0.1% trifluoroacetic acid / distilled water) was flowed at 0 ° C. at a flow rate of 8 ml / min to equilibrate. The above (2
-2) The lyophilized product obtained in 2) was added to 1 M
And 10 ml of each chromatography was performed four times. That is, 10 ml of an acetic acid solution of a lyophilized product was applied to the column, and the solution A (0.1% trifluoroacetic acid / distilled water) volume 80% / solution B (0. 1% trifluoroacetic acid / 100
% Acetonitrile) from 20% capacity to solution A (0.1%
Trifluoroacetic acid / distilled water) volume 40% / solution B (0.1
% Trifluoroacetic acid / 100% acetonitrile) with a linear gradient up to a volume of 60%. The eluate was used for fraction No. 8 ml each. , The mixture was dispensed into 1 ml portions, and concentrated and dried using a vacuum concentrator (Servant). To this dried product, 0.03 ml of dimethyl sulfoxide was added and dissolved, and GALR1 and GAL1 were dissolved.
An assay sample for measuring the R2 activating effect was used. (2-4) GALR2 by [ ³⁵ S] GTPγS binding test
Isolation and Purification of Compound Activating (Galanin Receptor Type 2) The assay sample obtained in (2-3) above is used for the GALR1 or GALR2 membrane fraction described in (1-3) above. [ ³⁵ S] GTPγS binding test
The receptor activating effect was measured. In the [ ³⁵ S] GTPγS binding test using the cell membrane fraction expressing GALR1, fraction No. Nos. 36-39 and Nos. 36-39. [ ^35]
S] GTPγS binding promoting activity was observed.
In the [ ³⁵ S] GTPγS binding test using the cell membrane fraction expressing R2, fraction No. 36-39; 42-4
3, and No. 3 [ ³⁵ S] GTPγS binding promoting activity was observed in 46-49 (FIG. 2). Further, the assay sample was diluted 100-fold with dimethyl sulfoxide, and 3 ml of the diluted sample was analyzed using a pig galanin radioimmunoassay kit (Peninsula). Galanin immunoreactivity was detected at 36-38 (FIG. 3). From the above results, fraction No.
It was determined that the component having a GALR1 and GALR2 activating action of 36-39 was porcine galanin. On the other hand, the fraction No. It was expected that the component having a GALR2 activating action of 46-49 was a compound different from galanin.

(2-5) Measurement of molecular weight of compound activating GALR2 (galanin receptor type 2)
The molecular weight of the GALR2 activating component (fraction Nos. 46-49) obtained in (2-4) was measured. Fraction No. 0.02 of the sample mixture of 46-49
0.08 ml of 0.1% trifluoroacetic acid / distilled water
, And 0.05 ml thereof was applied to a G2000SWXL (Tosoh Corporation) column, and 0.1% trifluoroacetic acid /
10% acetonitrile was flowed at a flow rate of 0.5 ml / min, and 0.25 ml of each eluate was collected (every 0.5 minutes). The fractions collected are concentrated and dried (SpeedVac Plus
SC210A; Savant), and directly dissolved in the suspension of the membrane fraction, and then treated with 50 nM [ ³⁵ S] GTPγS (New England Nucleic Acid).
was added, and the [ ³⁵ S] GTPγS binding amount was measured by the method described in (1-3) below. as a result,
The component having a GALR2 activating action ([ ³⁵ S] GTPγS binding promoting activity) was obtained from fraction Eluted at 35 (retention time 17-17.5 minutes) [FIG. 4]. A known peptide analyzed under the same conditions (Adrenomedu
lin, Gastric inhibitory peptide, PACAP38, Neuropep
tide Y, β-endorphine, Galanin, SRIF28 (Somatostati
n 28), Bovine Serum Albumin, Trypsin inhibitor, Ly
sozyme) (FIG. 5). The molecular weight of the component having GALR2 activating activity of 46-49 was estimated to be about 5,000 to about 7,000.
On the other hand, the molecular weight of known porcine galanin (Peptide Research Institute) is 3157.4 (retention time 18.858).
Minute). Therefore, fraction No. The compound having a GALR2 activating effect obtained as 46-49 is
It was suggested that the substance was different from known galanin.

Reference Example 3 Purification of Compound Having GALR2 (Galanin Receptor Type 2) Activating Action (3-1) Preparation of Pig Hypothalamic Extract The above-mentioned (2) ―
The method of 1) was simplified as follows. Frozen swine hypothalamus (Tokyo Shibaura Organ Co., Ltd.) was sliced into thin slices using a kitchen knife (about 1 kg) and boiled 2500
In a 5000 ml beaker containing 5 ml of distilled water, add 10 ml
Boil for a minute. Water bath of the boiled hypothalamus with beaker,
Then, the temperature was lowered to about 4 ° C. in an ice bath, and the hypothalamus was homogenized with distilled water used for boiling for 10 minutes using a Polytron homogenizer. To the obtained homogenate, 150 ml of acetic acid and 8 ml of 6N hydrochloric acid were added dropwise to make the final concentrations thereof 1 M and 20 mM, respectively, and the mixture was stirred overnight (about 16 hours) using a stirring blade. The homogenate was centrifuged at 8000 rpm for 30 minutes using a high-speed centrifuge (CR26H, Hitachi RR10A rotor: Hitachi, Ltd.), and the obtained supernatant was filtered with a gauze to remove lipid fragments. By repeating the above operation four times, about 4
An extract was prepared from the kg hypothalamus.

(3-2) Concentration and crude fractionation of porcine hypothalamus extract (3-2-1) Purification by octadodecyl reversed-phase chromatography Silica gel having octadodecyl groups immobilized thereon (YMC, OMC)
DS-AM 120-S50) after swelling with methanol, 5cm in diameter
Was packed to a volume of 400 ml and equilibrated with 1 M acetic acid. In this column (3-1)
After impregnating half the amount of the extract prepared in (2 kg for the hypothalamus) at a flow rate of 400 ml / h, at a flow rate of 400 ml / h,
About 1000 ml of 1 M acetic acid, then about 1200 ml of 2
The gel was washed by running 0% acetonitrile / 0.1% trifluoroacetic acid. Finally, at a flow rate of 400 ml / h,
About 2000 ml of 60% acetonitrile / 0.1% trifluoroacetic acid was applied to the column to elute the crude peptide fraction of interest. The obtained eluate was concentrated using an evaporator, and then lyophilized with a freeze dryer (12EL; VirTis). Perform the above operation twice, about 4kg
A lyophilized powder of (200) extracts of the hypothalamus was prepared. (3-2-2) Purification by SP-Sephadex ion exchange chromatography SP-Sephadex C25 (Pharmacia Biotech) swollen in 10 mM hydrochloric acid was placed on a glass column having a diameter of 5 cm.
The solution was filled to a volume of 120 ml, washed with 100 mM hydrochloric acid, and then equilibrated with 1 M acetic acid. (3-2-1)
Lyophilized powder of the hypothalamus extract obtained by
SP-Sephadex dissolved in 00 ml of 1 M acetic acid and equilibrated
The sample was applied to a C25 column at a flow rate of 400 ml / h. About 600
ml of 1M acetic acid, about 600 ml of 1M pyridine, about 6
00 ml of 1M pyridine / acetic acid (pH 5.0) was sequentially passed through the column at a flow rate of 400 ml / h.
This was dispensed into 0.2 ml each, and concentrated and dried with a vacuum concentrator (Servant). 0.04 ml of dimethyl sulfoxide was added to and dissolved in the dried product,
The GALR2 activating effect was measured by the test method described in the above (1-3). As a result, the target component having a GALR2 activating action is 1M pyridine / acetic acid (pH 5.
0). From these results, it was considered that the component (compound) having the GALR2 activating action was a strongly basic substance. (3-2-2) Purification by Sephadex G50 Gel Filtration Chromatography The 1M pyridine / acetic acid (pH 5.0) eluate fraction obtained in the above (3-2-2) was made up to 100 ml using an evaporator. S concentrated and equilibrated with 1 M acetic acid
ephadex G50 (Pharmacia Biotech) column (diameter 6
cm, capacity 3000 ml) at a flow rate of 400 ml / h, and then 1M acetic acid was passed through the column at a flow rate of 400 ml / h, and 33 ml of the eluate was collected in fraction No. 1 each. , And the mixture was dispensed into 0.25 ml each and concentrated and dried with a vacuum concentrator (Savant). To this dried product, 0.03 ml of dimethyl sulfoxide was added and dissolved, and the GALR2 activating effect was measured by the test method described in the above (1-3). As a result, the target component having the GALR2 activating action is mainly composed of fractionated fraction No. 66-
80 was found to be eluted. In addition, the fraction No. Nos. 55-65 and No. 5; Weak activity was also detected in 45-54. Fraction No. 66-80,5
5-65 and 45-54 were each mixed and lyophilized. (3-3) Purification of Compound Having GALR2 Activating Activity Using HPLC (3-3-1) Purification by TSKgel ODS80TM Reversed-Phase High-Performance Liquid Chromatography TSKgel ODS80TM Reversed-Phase High-Performance Liquid Chromatography Column (Tosoh, 22.5 mm Solution (0.1% trifluoroacetic acid / distilled water) at 40 ° C. at a flow rate of 4 ml / min to equilibrate. The above (3-2-
Each lyophilized product of the Sephadex G50 preparative fraction obtained in 3) was dissolved in 1 M acetic acid, and applied to the respective columns. / Distilled water) Capacity 67% / B
The eluate was recovered by increasing the volume of the solution (0.1% trifluoroacetic acid / 60% acetonitrile) from 33% to a volume of 100% solution B (0.1% trifluoroacetic acid / 60% acetonitrile) with a linear gradient from 100%. Eluate 8m
l fraction No. And attached. In addition, fraction No. The lyophilizate of 66-80 was chromatographed in two parts and fraction N
o. 55-65 and 45-54 were each separated by one chromatography. Using 4 μl of the fractionated fraction directly, the GALR2 activating effect was measured by the test method described in the above (1-3). As a result, three types of activity peaks were mainly observed in all the chromatographys. Fraction No. The elution position of the component having an activating effect observed in 35-38 almost coincided with the elution position of galanin described in Reference Example 2. In addition, the fraction No. 47-
The activating component found in No. 50 was GAL described in Reference Example 2.
The elution position almost coincided with the elution position of a component having an R2 activating effect (which did not activate GALR1 and was not detected by the aforementioned porcine galanin radioimmunoassay kit).
Therefore, fraction No. GAL using 47-50
Purification of the component having the R2 activating action was to be performed, and these fractions were mixed and freeze-dried. (3--3-2) Purification by TSKgel CM-2SW ion-exchange high-performance liquid chromatography A column for TSKgel CM-2SW ion-exchange high-performance liquid chromatography (Tosoh, 0.46 cm × 25 cm) was used for 20
Solution A (10 mM ammonium formate / 40% acetonitrile) at a flow rate of 1 ml / mi at a flow rate of 1 ml / min.
n and equilibrated. The lyophilized product of the preparative fraction of reversed-phase high-performance liquid chromatography obtained in (3-3-1) was dissolved in solution A, applied to the column, and then subjected to solution A at a flow rate of 1 ml / min for 60 minutes ( The eluate was recovered by increasing the linear gradient from 100% volume of 10 mM ammonium formate / 40% acetonitrile) to 100% volume of solution B (500 mM ammonium formate / 40% acetonitrile). The eluate was collected in fractions of 0.5 ml each. And attached. Using 1 μl of the fractionated fraction directly,
The GALR2 activating action was measured by the test method of -3). As a result, the main activity peak was the fraction No. 8
9-92. These fractions were combined and used for subsequent purification.

(3-3-3) Purification by Diphenyl Reversed-Phase High-Performance Liquid Chromatography A column for diphenyl reversed-phase high-performance liquid chromatography (Vydak 219TP54, 0.46 cm × 25 cm) was subjected to
Solution A (0.1% trifluoroacetic acid / distilled water) was flowed at a flow rate of 1 ml / min at ℃ to equilibrate. The above (3-3
After directly applying the preparative fraction of the ion exchange high performance liquid chromatography obtained in -2) to the column, the flow rate was 1
From 100% solution A (0.1% trifluoroacetic acid / distilled water) volume to 100% solution B (0.1% trifluoroacetic acid / 60% acetonitrile) volume in 50 ml / min for 1 minute, Then, at a flow rate of 1 ml / min,
The eluate was recovered by increasing the concentration of solution B to 100% in a linear gradient over 0 minutes. The eluate was used for fraction No. 1 ml each. And attached. Using 1 μl of the fractionated fraction directly, according to the test method of (1-3) above,
GALR2 activating effect was measured. As a result, the main activity peak was the fraction No. 24-26.
These fractions were combined and used for subsequent purification. (3-3-4) Purification by Super Phenyl Reversed-Phase High-Performance Liquid Chromatography TSKgel Super Phenyl Reversed-Phase High-Performance Liquid Chromatography Column (Tosoh, 0.46 cm × 10 cm)
Solution A (0.1% trifluoroacetic acid / distilled water) was flowed at a flow rate of 1 ml / min at ℃ to equilibrate. (3-3-
The diphenyl reverse phase high performance liquid chromatography preparative fraction obtained in 3) was directly applied to the column, and then the solution A (0.1% trifluoroacetic acid / distilled water) was removed from 100% by volume at a flow rate of 1 ml / min for 1 minute. Solution B (0.1% trifluoroacetic acid / 60% acetonitrile) is rapidly raised to a volume of 45%, which is then added at a flow rate of 1 ml / min to 8%.
The eluate was recovered by raising the concentration of solution B to 55% in a linear gradient over 0 minutes. The eluate was used for fraction No. 1 ml each. And attached. Using 1 μl of the fractionated fraction directly, G
ALR2 activating effect was measured. As a result, the main activity peak was the fraction No. Nos. 44-45 and Nos. 50
It was detected at -52. Fraction No. 44-45 were mixed and re-chromatography was performed under the same conditions, and 0.5 ml of each eluted fraction was collected. On the other hand, No. 50, 51 and 52 were separately subjected to re-chromatography under the same conditions,
The eluted fraction was fractionated. Using 2 μl of each preparative fraction directly, according to the test method of (1-3) above,
GALR2 activating effect was measured. As a result, no. 4
Fraction No. 4-45 obtained by the re-chromatography operation of No. 4-45. 93-95 and fraction Nos.
No. 50 obtained by the re-chromatography operation. An activity peak was detected at 105-107. Fraction No. No. 51 obtained by the re-chromatography operation. The main activity peak at No. 105-107 and the fraction No. A weak activity peak was detected at 108-109. Furthermore, the fraction No. 5
Fraction No. 2 obtained by the re-chromatography operation of No. 2 A major activity peak was broadly detected in 106-110.

(3-3-5) Purification by Super ODS reversed-phase high-performance liquid chromatography (3-3-5-1) Purification of GALR2-activated main component (3-3-5-1-1) Trifluoroacetic acid Chromatography in the presence of TSKgel Super ODS reversed-phase high-performance liquid chromatography column (Tosoh Corporation, 0.46 cm × 10 cm)
Solution A (0.1% trifluoroacetic acid / distilled water) was flowed at 40 ° C. at a flow rate of 1 ml / min to equilibrate. (3-3
-4) Super phenyl reversed-phase high-performance liquid chromatography fractionation fraction No. After the 93-95 was directly attached to the column, the solution A (0.1% trifluoroacetic acid / distilled water) volume was changed from 100% to the solution B (0.1% trifluoroacetic acid / 60%) at a flow rate of 1 ml / min for 1 minute. % Acetonitrile) volume was increased with a linear gradient to 50%, which was then increased at a flow rate of 1 ml / min over 84 minutes with a linear gradient to a 62.5% solution B concentration and the eluate was collected. The eluate was collected in fractions of 0.5 ml each. And attached. Preparative fraction 2μ
1 was directly used, and the GAL was determined by the test method (1-3).
The R2 activating effect was measured. As a result, the activity peak was the fraction No. 96-97. The obtained fraction No. 96-97 were mixed and re-chromatography was performed under the same conditions, and 0.5 ml fractions were collected. Although a single peak was detected by ultraviolet (UV) absorption at 210 nm, a shoulder of a peak suggesting contamination was found. Each preparative fraction 2
μl was directly used, and the test method described in (1-3) above was used.
The LR2 activating effect was measured. As a result, the activity peak was the fraction No. 98-100, which corresponded to the portion excluding the shoulder portion of the peak detected by ultraviolet absorption at 210 nm. In order to remove impurities contained in the shoulder portion of the peak, further purification was performed by the method described in (3-3-5-1-2). (3-3-5-1-2) Chromatography in the presence of heptafluorobutyric acid A column for TSKgel Super ODS reverse-phase high-performance liquid chromatography (Tosoh Corporation, 0.46 cm × 10 cm) was used.
Solution A (0.1% heptafluorobutyric acid / distilled water) was flowed at 40 ° C. at a flow rate of 1 ml / min to equilibrate. (3-
The fraction No. obtained in 3-5-1-1). 98-
100 was directly applied to the column, and the flow rate was 1 ml / mi.
n In 1 minute, the volume of solution A (0.1% heptafluorobutyric acid / distilled water) from 100% to the volume of solution B (0.1% heptafluorobutyric acid / 100% acetonitrile) was rapidly increased from 35% to 35%. Next, at a flow rate of 1 ml / min, the solution B concentration was raised to 50% in a linear gradient over a period of 60 minutes, and the eluate was collected. The eluate was collected in fractions of 0.5 ml each. And attached. Preparative fraction 2
μl was directly used, and the test method described in (1-3) above was used.
The LR2 activating effect was measured. As a result, the activity peak was the fraction No. 67-69. This activity peak completely coincided with the earlier-eluted ultraviolet absorption peak of the two ultraviolet absorption peaks at 210 nm, which was determined to have been purified to a single peptide. The component having this activating effect is called a GALR2 activating component. (3-3-5-2) Purification of GALR2 Activating Secondary Component (1) A column for TSKgel Super ODS reversed-phase high-performance liquid chromatography (Tosoh Corporation, 0.46 cm × 10 cm) was used.
Solution A (0.1% heptafluorobutyric acid / distilled water) was flowed at 40 ° C. at a flow rate of 1 ml / min to equilibrate. (3-
Super Phenyl Reversed-Phase High Performance Liquid Chromatography Preparative Fraction No. 3-4) 105-107 (from fraction No. 50), fraction no. 10
5-107 (from fraction No. 51), fraction no. 106-107 (from fraction No. 52) were mixed and directly applied to the column.
solution A (0.1% trifluoroacetic acid /
From 100% volume of distilled water), the volume of solution B (0.1% trifluoroacetic acid / 60% acetonitrile) was rapidly increased to 52.5%, which was then added at a flow rate of 1 ml / min.
The eluate was recovered by raising the concentration of solution B to 65% in a linear gradient over a period of minutes. The eluate was collected in fractions of 0.5 ml each. And attached. Using 2 μl of the fractionated fraction directly, according to the test method of (1-3) above,
GALR2 activating effect was measured. As a result, the activity peak was the fraction No. 75-76. The obtained fraction No. 75-76, and the same chromatography was performed again under the same conditions. And attached. The eluate is 2
It was detected as a single peak at 10 nm ultraviolet absorption. Using 3 μl of the fractionated fraction directly, the above (1-
The GALR2 activating action was measured by the test method 3). As a result, the activity peak was the fraction No. 76-
78 were detected. This activity peak coincided with the ultraviolet absorption peak at 210 nm, and it was determined that the active ingredient had been purified to a single peptide. The component having this activation action is referred to as GALR2 activation accessory component (1).

(3-3-5-3) Purification of GALR2 Activating Secondary Component (2) A column for TSKgel Super ODS reversed-phase high-performance liquid chromatography (Tosoh Corporation, 0.46 cm × 10 cm) was used.
Solution A (0.1% heptafluorobutyric acid / distilled water) was flowed at 40 ° C. at a flow rate of 1 ml / min to equilibrate. (3-
Super Phenyl Reversed-Phase High Performance Liquid Chromatography Preparative Fraction No. 3-4) 108-109 (from fraction No. 51), fraction no. 10
8-110 (from fraction No. 52),
After direct application to the column, the volume of solution A was rapidly increased from 100% volume to 100% solution B (0.1% trifluoroacetic acid / 60% acetonitrile) to 52.5% at a flow rate of 1 ml / min for 1 minute. At a flow rate of 1 ml / min.
The eluate was recovered by raising the concentration of solution B to 65% in a linear gradient over a period of minutes. The eluate was collected in fractions of 0.5 ml each. And attached. Using 2 μl of the fractionated fraction directly, according to the test method of (1-3) above,
GALR2 activating effect was measured. As a result, the activity peak was the fraction No. 79-80 detected. The obtained fraction No. 79-80 was mixed, and the same chromatography was performed again under the same conditions. When 0.5 ml of each fraction was collected, a single peak was detected by ultraviolet absorption at 2210 nm. Using 3 μl of the fractionated fraction directly, the GALR2 activating effect was measured by the test method described in the above (1-3). As a result, the activity peak was the fraction No. 79-81. This activity peak is 2
It coincided with the ultraviolet absorption peak at 10 nm, and it was determined that the active ingredient had been purified to a single peptide. The component having this activation action is referred to as GALR2 activation accessory component (2).

Reference Example 4 Analysis of Amino Acid Sequence (4-1) Analysis of N-terminal amino acid sequence of main component having GALR2 activating action Fraction N obtained in (3-3-5-1-2) above
o. The GALR2-activated main component of 67-69 was dissolved in 20 μl of 0.1% trifluoroacetic acid / 30% acetonitrile, dropped on a peptide support disk (Beckman), and dried with nitrogen gas. This is a protein sequencer (Beckman, LF3400D
T), an automatic Edman degradation reaction was performed by a standard analysis method (procedure 40), and amino acid sequence analysis was performed. The PTH amino acids identified for each cycle are shown in [Table 1].

[Table 1] From Table 1, the amino acid sequence up to the 34th residue from the N-terminus of the GALR2 activation main component could be determined as follows (residue X at the 28th position is unidentified). APVHRGRGGWTLNSAGYLLGPVLHPPSXAEGGGK (N-terminal amino acid sequence of main component for activating GALR2, SEQ ID NO: 11) A sequence consisting of 13 residues from the 9th to 21st residues from the N-terminus in the obtained amino acid sequence is a known pig galanin. N-terminal amino acid sequence. However, the amino acid sequence before and after that did not match any sequence of porcine galanin and its precursor. In addition, since a known peptide corresponding to the present amino acid sequence was not found, it was assumed that the obtained GALR2 activation main component was a novel peptide. (4-2) Subcomponent having GALR2 activating action (1)
N-terminal amino acid sequence analysis of the fraction No. obtained in the above (3-3-5-2)
The GALR2 activation accessory component of 76-78 was analyzed in the same manner as in (4-1). The amino acids identified for each cycle are shown in [Table 2].

[Table 2] From Table 2, the amino acid sequence from the N-terminus to the 32nd residue of the GALR2 activation subcomponent (1) could be determined as follows (residue X at the 28th residue is unidentified). . APVHRGRGGWTLNSAGYLLGPVLHPPSXAEGG (N-terminal sequence of GALR2 activation accessory component (1), SEQ ID NO: 12) This amino acid sequence is the GALR obtained in (4-1) above.
It completely corresponded to 32 residues of the N-terminal amino acid sequence of the two activated main components.

(4-3) N-terminal amino acid sequence analysis of subcomponent (2) having GALR2 activating action The fraction No. obtained in the above (3-3-5-3)
The 79-81 GALR2 activation accessory component (2) was
1% 0.1% trifluoroacetic acid / 30% acetonitrile, ammonium bicarbonate (final concentration 1%), TLC
K-chymotrypsin (final concentration 10 pmol, Sigma)
Then, distilled water was added to make the volume 50 μl, and the mixture was reacted at 37 ° C. for 1 hour. Spheri-5 RP-18 column for reversed-phase high-performance liquid chromatography (Brownley, 2.1 mm × 30
mm) was equilibrated at 25 ° C. by flowing solution A (0.1% trifluoroacetic acid) at a flow rate of 300 μl / min. After the entire amount of the enzyme reaction solution was applied to the column, the flow rate was 300 μl /
In 30 minutes, the volume of solution A (0.1% trifluoroacetic acid) from 100% to the volume of solution B (0.1% trifluoroacetic acid / 70% acetonitrile) is increased from 70% to 70% over 30 minutes, and the ultraviolet absorption at 210 nm is reduced. 4 elution peaks shown (CH
Y-1, CHY-2, CHY-3 and CHY-4) were collected as eluted fractions. The four fractions collected (digested chymotrypsin fragments) were concentrated under a nitrogen stream until the volume became 50 μl or less, and N-terminal amino acid sequence analysis was performed using a protein sequencer in the same manner as in (4-1). For CHY-3, Beckman LF3400DT protein sequencer was used.
For CHY-1, CHY-2 and CHY-4, Applied Biosystems 477A protein sequencer was used. The following sequence was obtained by N-terminal amino acid sequence analysis. CHY-1: APVHRGRGG (SEQ ID NO: 13) CHY-2: XAIDGLPYPQS (X is unidentified) (SEQ ID NO:
16) CHY-3: LLGPVLHPPSXAEGGGKTALGILDL (X is unidentified) (SEQ ID NO: 15) CHY-4: TLNSAG (SEQ ID NO: 14) The GALR2-activating main component obtained in the above (4-1) or the above (4-) Referring to the result of the N-terminal amino acid sequence of the GALR2 activation accessory component (1) obtained in 2),
It was inferred that the R2 activation accessory component (2) had the following N-terminal amino acid sequence. APVHRGRGGWTLNSAGYLLGPVLHPPSXAEGGGKTALGILDL (SEQ ID NO: 17)

Reference Example 5 Pig-type ligand peptide (1-
CDNA coding for 60) 1) Degenerated PCR (Degenerated PCR) method
Cloning of porcine GALR2 activator gene fragment using TRIZOL reagent (Gibco BRL) from pig whole brain
Total according to the method described in the attached manual
RNA was extracted. Then an oligo dT cellulose column (m
RNA Purification Kit, Pharmacia)
The total RNA according to the method described in the
From poly (A)⁺RNA was prepared. Furthermore, 3 'RACE System
 for rapid amplification of cDNA ends (Gibco BRL
Using the method described in the attached manual.
Therefore, the Poly (A)⁺First strand cDNA from RNA
Done. In addition, partial amino of porcine GALR2 activating component
Add the following degenerate primer designed based on the acid sequence.
Done. pGAL4-7F: 5'-CAYMGNGGIMGNGGIGGSTGGAC-3 '(SEQ ID NO: 20) pGAL9-3F: 5'-GGHTGGACNCTNAAYAGYGC-3' (SEQ ID NO: 21) pGAL34-1R: 5'-ATICCNAGIGCNGTYTTICCYTT-3 '(SEQ ID NO: 22) Using the first strand cDNA as a template, pGAL4-7F
And first-stage PC using pGAL34-1R as primer
An R reaction was performed. The reaction solution in the PCR reaction was Ta
0.5 μl of q polymerase (Takara Shuzo Co., Ltd.)
10x PCRbuffer (500 mM KCl-100 mM TrisHCl,
 pH 8.3) in 10 μl, 25 mM MgCl _Two6 μl, 2.
8 μl of 5 mM dNTP mixture, primers pGAL4-7F and
And primers pGAL34-1R (10 μM for each)
l, and the template cDNA (first strand cDN described above).
A) is prepared by mixing 8 μl of distilled water and 47.5 μl of distilled water.
Was. As the PCR, 1) Initial denaturation (94 ° C., 30 seconds)
2) 32 cycle reactions (94 ° C., 20 seconds-
3) Final elongation after 55 ° C for 30 seconds -72 ° C for 30 seconds)
Reaction (72 ° C., 4 minutes) was performed to obtain a first-stage PCR product.
Using the obtained first-stage PCR product as a template, the second-stage PCR
A nested PCR was performed. The reaction solution is Taq polymera
0.5 μl of se (Takara Shuzo Co., Ltd.) and the attached 10x PCR bu
ffer (500 mM KCl-100 mM Tris-HCl, pH 8.3)
1, 25 mM MgCl_Two6 μl, 2.5 mM dNTP mixtur
e, 8 μl, primers pGAL9-3F and pGAL34-1R (both
10 μl each, and 10 μl of the template cDNA (initial).
PCR product) and 50.5 μl of distilled water
It was produced. As the PCR, 1) initial denaturation (94 ° C.)
・ 30 seconds) 2) 32 cycle reactions (94 ° C. ・ 20
3 seconds after -55 ° C for 30 seconds and -72 ° C for 30 seconds).
Perform a final extension reaction (72 ° C, 10 minutes) to produce nested PCR.
I got something. Add 2.0% agarose to nested PCR products
Perform gel electrophoresis and stain with cyber green.
A piece of gel containing the 00 bp band was cut out with a razor. The
Gene Clean DNA Extraction Kit (BIO 101) from gel pieces
The DNA fragment which was a nested PCR product was recovered using the method. This
DNA fragment from TOPO TA Cloning Kit (Invitrogen)
Key and follow the method described in the attached manual.
Ligated to the plasmid vector pCRII attached to the kit
did. Transfer the resulting plasmid to QIAwell 8 Ultra plasmid
Purification was performed using a purification kit (QIAGEN). The
Antibodies for sequencing nested PCR products in rasmids
The Dye Terminator Cycle Sequencing Kit (Applied
 Biosystems, PerkinElmer)
Optical automatic sequencer (DNA sequencer Prism 377: A
nes using pplied Biosystems, PerkinElmer)
The nucleotide sequence of the ted PCR product was decoded. As a result, pig G
98b encoding a partial peptide of ALR2 activator
Obtaining several kinds of plasmids having p DNA fragment
Was completed. Degenerate primers were used in the PCR reaction
Therefore, the DNA in these several plasmids
A difference was found in the nucleotide sequence at the part corresponding to the immer. Less than
Below are two representative clones, pCR100-6 and pCR100-7.
Shows the nucleotide sequence of the nested PCR product. pCR100-6: GGCTGGACTT TAAATAGTGC TGGTTACCTC CTGGGTCCCG TACTCCATCC GCCCTCC
AGGGCTGAAGGAG GCGGGAAGGG CAAAACAGCC CTGGGCAT (SEQ ID NO: 23) pCR100-7: GGTTGGACTT TGAACAGTGC TGGTTACCTC CTGGGTCCCG TACTCCATCC GCCCTCC
AGGGCTGAAGGAG GCGGGAAGGG CAAAACCGCC CTAGGCAT (SEQ ID NO: 24) 2) Encoding porcine ligand peptide (1-60)
Cloning of cDNA Poly (A) obtained from porcine whole brain⁺ From RNA, ZAP-c
DNA Gigapack III Gold cloning kit (STRATAGENE)
Creates porcine whole brain cDNA phage library
did. The method followed the manual attached to the kit.
Obtained phage (2,200,000 plaque forming units)
E. coli XL1-BlueMRF '(STRATAGE
NE), and then infected with NZY medium agar plate 12
The seeds were spread on 0 sheets and cultured at 37 ° C. for 8 hours. E. coli obtained
Hybond-N + nylon membrane from plaque
(Amersham). This nylon membrane
0.5N sodium hydroxide containing 1.5M sodium chloride
Thorium solution, 0.5M with 1.5M sodium chloride
 Tris buffer solution (pH 7.0) and 2 × SSC solution
And then air-dried. Nylon membrane obtained
Ren was added to hybridization buffer (5 × SSPE,
5 × Denhardt's solution, 0.5% SDS, 0.1 mg / m
l salmon sperm DNA) at 60 ° C for 24 hours,
Then, a hybridization probe (5 × 10^Five
cpm / ml)
(Ditto), and kept at 60 ° C. for 24 hours. The hive
The redidation probe was constructed using the plasmid pCR100 described above.
Using a 1: 1 mixture of -6 and pCR100-7 as a template, the following 2
Primers pGAL9-3F and pGAL34-8R
DNA fragment was used. pGAL9-3F: 5'-GGHTGGACNCTNAAYAGYGC-3 '(SEQ ID NO: 25) pGAL34-8R: 5'-ATDCCBAGGGCDGTTTTGCCCTT-3' (SEQ ID NO: 26)
0.5 ml and the attached 10x Ex Taq buffer (20 mM
 MgCl_Twoincluding. ) With 5 μl and 2.5 mM dNTPmixture
4 μl, [α-³²P] dCTP (6000 Ci / mM)
10 μl, primers pGAL9-3F and pGAL34-8R (that
0.5 μl each and 10 μM each, and template cDNA
1 μl, and 29 μl of distilled water were mixed).
After initial denaturation at 6 ° C for 30 seconds, -55 ° C for 20 seconds at 94 ° C
32 cycles of 30 seconds at -72 ° C for 30 seconds,
And a final extension reaction at 72 ° C for 4 minutes to perform amplified DNA
A fragment was obtained. Niro after the hybridization reaction
0.2% SS containing 0.1% SDS
After washing with C buffer at 50 ° C. for 30 minutes, X-ray
Sensitized screen using film (Kodak, BioMax MS)
Autoradiography in the presence of exposure
(-70 ° C, 3 days). The autoradiography
Positive plaques detected from the results were isolated and
SM buffer containing 0.1 ml of LUM (100 mM NaCl, 8 mM
MgSO_Four, 0.01% gelatin, 50 mM Tris-HCl, pH7.5) 5
Phage was extracted in ml. The phage obtained is again
After infecting E. coli XL1-Blue MRF ', NZY medium agar
And cultivated at 37 ° C. for 8 hours. Got
Transfer phage from E. coli plaque to nylon membrane (A
Copy to Masham, Hybond-N +)
Hybridization was performed by the method. Autorati
A single positive clone was isolated by
Repeat the same series of operations as
A single phage clone was obtained.

Next, a plasmid was isolated and purified from the phage clone by the following method. The phage solution (1 μl) was added to E. coli XPORT (50 μl) and helper phage (1
0 μl), mixed with E. coli XLOLR (5 μl), NZ
It was spread on a Y agar plate and cultured at 37 ° C. for 8 hours. A small colony of Escherichia coli XLOLR generated in the Escherichia coli XPORT plaque was picked up with a toothpick and cultured overnight at 37 ° C. on an LB medium agar plate containing ampicillin and tetracycline. E. coli XLO from a single colony
LR was picked up and liquid cultured in LB medium.
After completion of the culture, Escherichia coli XLOLR was collected, and the plasmid was purified using a plasmid purification kit (Qiagen, QIAwell 8 Ultra). Pig GALR2 in the plasmid
The reaction for determining the nucleotide sequence of the cDNA encoding the activator is performed using the Dye Terminator Cycle Sequencing Kit (App
lied Biosystems, PerkinElmer) and a fluorescent automatic sequencer (DNA sequencer Prism).
377: Applied Biosystems, Perkin Elmer) to determine the nucleotide sequence of the cDNA encoding the porcine GALR2 activator. As a result, plasmid pGR2PL6 having a 974 bp DNA fragment encoding the full-length peptide of porcine GALR2 activator and plasmid pGR2PL3 having a 1007 bp DNA fragment could be obtained. GALR2 encoded by both DNA sequences
Although significant differences were observed in the chain length and amino acid sequence of the activator precursor, the sequence of the mature activator (mature peptide) portion expected to be produced by processing was identical. The resulting two plasmids were introduced into Escherichia coli TOP10 by a known method, and the transformants were Escherichia coli TOP10 / pGR2PL6 and Escherichia coli, respectively.
li TOP10 / pGR2PL3 was obtained.

Reference Example 6 Pig-type ligand peptide (1-
60): Ala-Pro-Val-His-Arg-Gly-Arg-Gly-Gly-Trp-Th
r-Leu-Asn-Ser-Ala-Gly-Tyr-Leu-Leu-Gly-Pro-Val-Leu-
His-Pro-Pro-Ser-Arg-Ala-Glu-Gly-Gly-Gly-Lys-Gly-Ly
s-Thr-Ala-Leu-Gly-Ile-Leu-Asp-Leu-Trp-Lys-Ala-Ile-
Asp-Gly-Leu-Pro-Tyr-Pro-Gln-Ser-Gln-Leu-Ala-Ser
Production of (SEQ ID NO: 31) Commercially available Boc-Ser (Bzl) 1-OCH ₂ -PAM resin (0.73 mM / g resin)
0.5 mM was placed in a reaction vessel of a peptide synthesizer ABI 430A, and Boc-Ala, Boc-Leu, Boc-Gln, Boc-Ser (Bzl), Boc-Pr were synthesized by the Boc-strategy (NMP-HOBt) peptide synthesis method.
o, Boc-Tyr (Br-Z), Boc-Gly, Boc-Asp (OcHex), Boc-Il
e, Boc-Lys (Cl-Z), Boc-Trp (CHO), Boc-Thr (Bzl), Boc-
Glu (OcHex), Boc-Arg (Tos), Boc-His (Bom), Boc-Val, B
oc-Asn was introduced in the desired amino acid sequence in order to obtain a desired protected peptide resin. After stirring 0.105 g of this resin together with 1.1 g of p-cresol and 1.2 ml of 1,4-butanedithiol in 10 ml of anhydrous hydrogen fluoride at 0 ° C. for 60 minutes, hydrogen fluoride was distilled off under reduced pressure. Diethyl ether was added to the product, and the precipitate was filtered. 50% aqueous acetic acid was added to the precipitate for extraction, the insoluble portion was removed, and the extract was concentrated sufficiently.
Subjected to Sephadex ^TM G-25 column packed with 0% aqueous acetic acid (2.0 × 80 cm), reverse phase chromatography column packed expanded, the LiChroprep ^TM RP-18 collected main fraction with the same solvent (2. 6 x 60cm) 0.1% TFA water
Wash with 200 ml, 300 ml of 0.1% TFA water and 300 m of 50% acetonitrile water containing 0.1% TFA
The main fractions were collected and concentrated by linear gradient elution using
Lyophilization yielded 39 mg of a white powder. This is further CM-
A linear gradient elution from 50 mM to 200 mM ammonium acetate was applied to an ion exchange chromatography column packed with Cellulofine ^™ , and the main fraction was collected and freeze-dried repeatedly to obtain 10 mg of a white powder. (M + H) by mass spectrometry ⁺ 6204.7 (calculated value 6205.2) HPLC elution time 21.8 minutes Column conditions Column Wakosil 5C18T 4.6 x 100mm Eluent: A solution-0.1% TFA water, B solution-0.1% TFA containing acetonitrile containing A / B: Linear gradient elution from 95/5 to 45/55 (25 min) Flow rate: 1.0 ml / min

Reference Example 7 Rat-type ligand peptide (1
-60): Ala-Pro-Ala-His-Arg-Gly-Arg-Gly-Gly-Trp-
Thr-Leu-Asn-Ser-Ala-Gly-Tyr-Leu-Leu-Gly-Pro-Val-Le
u-His-Leu-Ser-Ser-Lys-Ala-Asn-Gln-Gly-Arg-Lys-Thr-
Asp-Ser-Ala-Leu-Glu-Ile-Leu-Asp-Leu-Trp-Lys-Ala-Il
e-Asp-Gly-Leu-Pro-Tyr-Ser-Arg-Ser-Pro-Arg-Met-Thr
(SEQ ID NO: 33) and human ligand polypeptide (1-60): Ala-Pro-Ala-His-Arg-Gly-Arg-Gly-Gly
-Trp-Thr-Leu-Asn-Ser-Ala-Gly-Tyr-Leu-Leu-Gly-Pro-V
al-Leu-His-Leu-Pro-Gln-Met-Gly-Asp-Gln-Asp-Gly-Lys
-Arg-Glu-Thr-Ala-Leu-Glu-Ile-Leu-Asp-Leu-Trp-Lys-A
la-Ile-Asp-Gly-Leu-Pro-Tyr-Ser-His-Pro-Pro-Gln-Pro
Production of -Ser (SEQ ID NO: 34) It can be obtained by performing synthesis and purification in the same manner as in Reference Example 1 using a commercially available Boc-Thr (Bzl) 1-OCH ₂ -PAM resin.

Reference Example 8 Antigen peptide used for preparation of monoclonal antibody Ala-Pro-Ala-His-Arg-Gly-Arg-Gly-Gly-
Production of Cys-NH ₂ (C-terminal amide of peptide having the amino acid sequence represented by SEQ ID NO: 44) Boc-Cys as in Reference Example 1 using commercially available p-methyl MBH resin (0.77 mM / g resin) (MeBzl), Boc-Gly, Boc-Arg (T
os), Boc-His (Bom), Boc-Ala, and Boc-Pro were condensed in the desired amino acid sequence, treated with hydrogen fluoride, and packed with 50% aqueous acetic acid. Sephadex ^™ G-25 column (2.0 × 80
cm) and freeze-dried to obtain 50 mg of a white powder. Mass analysis (M ⁺ H) + 980.5 (calcd 980.5) HPLC elution time 5.2 minutes Column conditions Column Wakosil 5C18T 4.6 x 100mm Eluent: A solution -0.1% TFA water, B solution -0.1% T
Using FA-containing acetonitrile, B solution concentration 0 to 50%
Linear gradient elution (25 min) Flow rate: 1.0 ml / min

Reference Example 9 Ala-Pro-Ala-His-Arg-Gly-Arg-
Preparation of Immunogen Containing Gly-Gly (SEQ ID NO: 35) Ala-Pro-Ala-His-Arg-Gly-Arg- obtained in Reference Example 8 above.
A complex of the peptide represented by Gly-Gly-Cys-NH ₂ (C-terminal amide of the peptide having the amino acid sequence represented by SEQ ID NO: 44) and Keyhole Limpet Hemocyanin (KLH) was prepared, and an immunogen was produced. did. That is, KLH 20 mg
Was dissolved in 1.4 ml of 0.1 M phosphate buffer (pH 6.5), and DM containing 2.2 mg (8 μmol) of N- (γ-maleimidobutyryloxy) succinimide (GMBS) was dissolved.
The mixture was mixed with 100 μl of the F solution and reacted at room temperature for 40 minutes.
After the reaction, the mixture was fractionated on a Sephadex G-25 column, and KLH (15 mg) into which a maleimide group was introduced and Ala-Pr
o-Ala-His-Arg-Gly-Arg-Gly-Gly-Cys-NH ₂ (SEQ ID NO:
(C-terminal amide form of a peptide having an amino acid sequence represented by No. 44), and 1.6 mg of the peptide described above.
The reaction was carried out at 2 ° C. for 2 days. After the reaction, 4
It was dialyzed for 2 days at ° C.

REFERENCE EXAMPLE 10 Immunization The immunogen Ala-Pro-Ala-His-Arg-Gly-Arg-Gly-Gly-Cys (-N
About 30 μg / animal of H ₂ ) -KLH complex was subcutaneously immunized with complete Freund's adjuvant. Thereafter, every three weeks, the same amount of the immunogen was boosted twice with incomplete Freund's adjuvant.

Reference Example 11 Preparation of enzyme-labeled antigen Horseradish peroxidase (HRP) -labeled Ala-Pro-
Ala-His-Arg-Gly-Arg-Gly-Gly-Cys-NH ₂ (SEQ ID NO: 4
Preparation of the peptide represented by the formula (4): Ala-Pro-Ala-His-Arg-Gly-Arg-Gly-Gly-Cys-NH obtained in Reference Example 8 above
₂ (C-terminal amide of the peptide having the amino acid sequence represented by SEQ ID NO: 44) and HRP
(For Enzyme-linked immunosorbent assay, manufactured by Boehringer Mannheim) to give a labeled product for enzyme-linked immunosorbent assay (EIA).
That is, HRP 6 mg (150 nmol) was added to 0.95
dissolved in 0.1 ml phosphate buffer, pH 6.5;
It was mixed with 50 μl of a DMF solution containing 0.42 mg (1.5 μmol) of MBS, reacted at room temperature for 30 minutes, and then fractionated on a Sephadex G-25 column. HRP 4.2 mg into which a maleimide group was introduced, thus produced.
(105 nmol) and Ala-Pro-Al prepared in Reference Example 9
a-His-Arg-Gly-Arg-Gly-Gly-Cys-NH ₂ (SEQ ID NO: 44
0.31 mg (315 nmo) of the peptide represented by the C-terminal amide form of the peptide having the amino acid sequence represented by
1) and reacted at 4 ° C. for 1 day. After the reaction, fractionation was carried out using an Ultrogel AcA44 (manufactured by LKB-Pharmacia) column, and HRP-labeled Ala-Pro-Ala-His-Arg-Gly-Arg-Gl.
y-Gly-Cys-NH ₂ was obtained.

Reference Example 12 Ala-Pro-Ala-His-Arg-Gly-Ar
Measurement of antibody titer in antiserum of mouse immunized with g-Gly-Gly-Cys (-NH ₂ ) -KLH complex Ala-Pro-Ala-His-Arg-Gly-Arg obtained in Reference Example 9 above -
The antibody titer in the mouse antiserum immunized with the Gly-Gly-Cys (-NH ₂ ) -KLH complex was measured by the following method. To prepare an anti-mouse immunoglobulin antibody-bound microplate, first, a 0.1 M carbonate buffer, pH 9.6 solution containing 100 μg / ml of anti-mouse immunoglobulin antibody (IgG fraction, manufactured by Kappel) was placed in a 96-well microplate. Each 100 μl was dispensed and left at 4 ° C. for 24 hours.
Next, the plate was washed with phosphate buffered saline (PBS, pH
After washing in 7.4), 300 μl of PBS containing 25% Block Ace (manufactured by Snow Brand Milk Products Co., Ltd.) was dispensed in order to cover excess binding sites in the wells, and at least 24 ° C. at 4 ° C.
Time processed. Buffer C [1% B] was added to each well of the anti-mouse immunoglobulin antibody-bound microplate.
Ala-Pro-Ala-His-Arg-Gly-Arg-Gl diluted with 50 μl of 0.02 M phosphate buffer, pH 7.0] containing SA, 0.4 M NaCl, and 2 mM EDTA, buffer C
100 μl of y-Gly-Cys (—NH ₂ ) -KLH complex antiserum was added and reacted at 4 ° C. for 16 hours. Next, the plate is
After washing with BS, the HRP prepared in Reference Example 11 above was used.
Labeled Ala-Pro-Ala-His-Arg-Gly-Arg-Gly-Gly-Cys-NH ₂
100 μl (diluted 500-fold with buffer C) was added and reacted at room temperature for 1 day. Next, after the plate was washed with PBS, the enzyme activity on the solid phase was measured using a TMB microwell peroxidase substrate system (KIRKEGAARD & PERRY LAB, IN).
C, handling funakoshi) was measured by adding 100 μl and reacting at room temperature for 10 minutes. Reaction was performed with 1M phosphoric acid 1
After stopping by adding 00 μl, the absorbance at 450 nm was measured with a plate reader (BICHROMATIC, manufactured by Dainippon Pharmaceutical Co., Ltd.). The results are shown in FIG. Immunized 8
Ala-Pro-Ala-His-Arg-Gly-Arg-Gly-Gl
y-Cys (-NH ₂₎ -KLH rise in antibody titer to complex was observed.

Reference Example 13 Rat-type ligand peptide
(1-60) Monoclonal antibody of (SEQ ID NO: 33)
Preparation of mice having a relatively high antibody titer
Dissolve 0 μg of immunogen in 0.25-0.3 ml of physiological saline
The final immunity is obtained by inoculating the solution
Done. 3-4 days after final immunization, spleen is removed from mouse
Squeezed and filtered with stainless steel mesh,
Floating on Minimum Essential Medium (MEM)
Then, a spleen cell suspension was obtained. Cells used for cell fusion
And BALB / C mouse-derived myeloma cells P3-X
63. Ag8. U1 (P3U1)
Pix in microbiology and im
Noology, 81, 1 (1978)]. Cell fusion is performed using the original method [
Char, 256, 495 (1975)]. Sand
That is, spleen cells and P3U1 each contained no serum.
Wash three times with fresh MEM and determine the ratio of spleen cells to P3U1 number.
Mix at 10: 1 and run at 800 rpm for 15 minutes
The heart was performed to precipitate the cells. The supernatant was sufficiently removed
Afterwards, loosen the precipitate lightly and use 45% polyethylene glycol.
0.3 ml of (PEG) 6000 (manufactured by Kochlight)
Then, fusion was carried out by allowing to stand in a 37 ° C. hot water bath for 7 minutes.
After fusion, MEM was added to the cells at a rate of 2 ml / min, for a total of 1 ml.
After adding 5 ml of MEM, centrifuge at 600 rpm for 15 minutes.
Qing was removed. The cell sediment contains 10% fetal bovine serum.
GIT Medium (Wako Pure Chemical) (GIT-10%
 FCS) P3U1 2 × 10 per ml^FiveI will be individual
Floating, 24-hole multi-dish (Limbro)
Was seeded in 192 wells at 1 ml / well. Sowing
Later, the cells were placed in a 5% CO 2 incubator at 37 ° C.
Cultured. HAT after 24 hours (hypoxanthine 1 × 10
^-FourM, aminopterin 4 × 10^-7M, thymidine 1.6 ×
10 ^-3M) containing GIT-10% FCS medium (HA
T medium) is added at 1 ml per well.
HAT selection culture was started. HAT selective culture
3, 6, and 9 days after the start of feeding, 1 ml of the old solution is discarded, and then 1 m
Continued by adding 1 HAT medium. Hive
The growth of the lidoma was observed 9 to 14 days after the cell fusion,
When the culture solution turns yellow (about 1 × 10⁶Cells / ml), supernatant
And measuring the antibody titer according to the method described in Reference Example 5.
did. Ala-Pro-Ala-His-Arg-Gly-Arg-Gly-Gly-Cys (-NH
_Two2.) Hybrids derived from mice immunized with -KLH complex
Mouse No. 8 as a typical example of
The results obtained using (see FIG. 7) are shown in FIG.
Was. Next, the hybridomas showing these high antibody titers
Was subjected to cloning by the limiting dilution method. Clonin
BALB / C mouse as feeder cells
5 x 10 cells per well^FiveAdd
I got it. After cloning, hybridomas are
Mice given 0.5 ml of mineral oil intraperitoneally
(BALB / C) is 1-3 × 10⁶Cells / animal intraperitoneally
6 to 20 days after the administration, antibody-containing ascites was collected.
Monoclonal antibody was isolated from the obtained ascites by protein-A
Purified by column. That is, 6-20 ml of ascites is equal
Binding buffer (3.5 M NaCl, 0.05% Na)
N_ThreeWith 1.5 M glycine, pH 9.0)
After that, the recombinant previously equilibrated with the binding buffer
Top protein-A-agarose (Repligen) column
And the specific antibody was eluted with an elution buffer (0.05% NaN)._ThreeTo
Elution with 0.1M citrate buffer, pH 3.0)
Was. The eluate was dialyzed against PBS at 4 ° C for 2 days.
And removed with a 0.22 μm filter (Millipore).
The bacteria were filtered and stored at 4 ° C or -80 ° C. Monochrome
When determining the class and subclass of a null antibody,
Enzyme-phosphorus using monoclonal antibody binding solid phase
Performed immunosorbent assay (ELISA)
Was. That is, 0.1 M carbonate buffer containing 2 μg / ml of antibody
Buffer solution, pH 9.6 solution in 96-well microplate
Each 100 μl was dispensed and left at 4 ° C. for 24 hours. the above
Excess binding of wells according to the method described in Reference Example 12.
After blocking the area with Block Ace, isotype
Mouse-TyperTM Sub-Isotyping Kit
Immobilized by ELISA using IORAD
I checked body class and subclass.

Reference Example 14 Competition Method Enzyme Immunoassay (Competition Method-EIA) Ala-Pro-Ala-His-Arg-Gly-Arg-Gly-Gly-Cys (-NH ₂ )-
The reaction specificity of the monoclonal antibody prepared using the KLH complex as an immunogen was examined by the following method. First, the antibody titer of each monoclonal antibody solution was examined by the method described in Reference Example 12, and the antibody concentration used in the competition method-EIA was determined as the antibody concentration (about 30%) at which the binding amount of the label became about 50% of the saturated binding amount. ５０50 ng / ml). Next, buffer C was added to the anti-mouse immunoglobulin antibody-bound microplate described in Reference Example 12 to the determined concentration.
50 μl of the antibody solution diluted with Ala-Pro-Ala-His-Arg-
Gly-Arg-Gly-Gly-Cys-NH ₂ (amide of SEQ ID NO: 44), porcine ligand peptide (1-60) (SEQ ID NO: 31) and rat galanin (SEQ ID NO: 61)
50% of buffer C solution containing 0.05% CHAPS
μl and the HRP-labeled Ala- described in Reference Example 11 above.
50 μl of Pro-Ala-His-Arg-Gly-Arg-Gly-Gly-Cys-NH ₂ (diluted 250-fold with buffer C) was added and reacted at 4 ° C. for 16 hours. After the reaction, the plate was washed with PBS and the enzyme activity on the solid phase was measured by the method described in Reference Example 12 above.
Table 3 shows the results.

[Table 3] As a typical example, a porcine ligand peptide (1-60)
The results of the competition method-EIA of the monoclonal antibody GR2-1N (IgG _2b , κ) showing the highest reactivity with (SEQ ID NO: 31) are shown in FIG. 9. GR2-1N is C
ys-Ala-Pro-Ala-His-Arg-Gly-Arg-Gly-Gly (SEQ ID NO:
44), it has the same degree of reactivity with the porcine ligand peptide (1-60) (SEQ ID NO: 31), and does not react with rat galanin (SEQ ID NO: 61).
GR2-1N porcine ligand peptide (1-60)
From the standard curve of (SEQ ID NO: 31), (B / B ₀ ) =
Pig-type ligand peptide giving 0.5 (1-60)
(SEQ ID NO: 31) The concentration was 6 nM, 4.95 ng / we.
ll and its detection sensitivity is about 0.1 nM [(B /
B ₀ ) = 0.8]. Therefore,
The competition method EIA using GR2-1N was carried out according to SEQ ID NO: 61.
(Rat galanin) without cross-reactivity with Ala-Pro-
Rat-type ligand peptide having Ala-His-Arg-Gly-Arg-Gly-Gly (SEQ ID NO: 33), pig-type ligand peptide (1-60) (SEQ ID NO: 31) or human-type ligand peptide (SEQ ID NO: 44) can be specifically measured up to about 0.1 nM.

Reference Example 15 Pig-type ligand peptide (1
-34) Preparation of (SEQ ID NO: 11) 5 nmol of porcine ligand peptide (1-60) was 1
50 pmol of lysyl endopeptidase (manufactured by Wako Pure Chemical Industries, Ltd.) in a reaction solution of 250 μl of 1% ammonium bicarbonate containing 6% acetonitrile was incubated at 37 ° C. for 3.5 hours. Spheri-5 RP-18 column for reversed-phase high-performance liquid chromatography (Brownley, 2.1 mm × 30
mm), solution A (0.1% trifluoroacetic acid) is flowed at a flow rate of 300 μl / min in advance and equilibrated at 25 ° C. After driving the enzyme reaction solution into the column, the flow rate was 300
While maintaining the μl / min, the solution B (0.
The concentration (1% trifluoroacetic acid / 70% acetonitrile) is increased to 70%. The eluate was monitored by absorbance at 210 nm, and the peak was manually collected. The peak fraction eluted at 20.2 minutes was M + H ⁺ 346 by mass spectrometry.
6.5, which was also confirmed by amino acid analysis to be the fragment ligand peptide (1-34) of the porcine ligand peptide (1-60).

Reference Example 16 Pig-type ligand peptide (1
-30) Preparation of (SEQ ID NO: 43) 5 nmol of porcine ligand peptide (1-60) was 8
50 μmol of endoproteinase Glu-C (Boehringer Mannheim) in a reaction solution of 250 μl of 1% ammonium bicarbonate containing 5% acetonitrile and 3%
The mixture was kept at 7 ° C for 6 hours and then at 25 ° C for 13 hours. The enzyme reaction solution was separated under the same conditions as in the case of the pig-type ligand peptide (1-34). The peak fraction eluted at 19.8 minutes gave M + H ⁺ 3167.2 by mass spectrometry,
Amino acid analysis also revealed that the porcine ligand peptide (1-6)
It was confirmed to be the fragment ligand peptide (1-30) of 0).

Reference Example 17 Galanin Receptor Binding Experiment Using [ ¹²⁵ I] Rat Galanin The membrane fraction prepared in the above (1-2) was used at a concentration of 13 μg /
ml (for the GALR1 membrane fraction) or 15 μg / ml
0.1% bovine serum albumin (BSA), 5 mM MgCl ₂ , 0.
5 mM phenylmethylsulfonyl fluoride, 2
The suspension was suspended in 50 mM Tris buffer (pH 7.3) containing 0 μg / ml leupeptin, 10 μg / ml pepstatin, and 8 μg / ml E-64, and 0.1 ml of each suspension was dispensed into small polypropylene test tubes (Falcon 2053). 5 nM [ ¹²⁵ I] rat galanin (New En
gland Nuclear) at 4 μl and variable concentration (100 μM)
To 100 pM dimethyl sulfoxide solution) of the peptide synthesis product of the present invention (porcine ligand peptide (1-6)
0) (SEQ ID NO: 31), a limited digest of the peptide synthesis product of the present invention (porcine ligand peptide (1-30) (SEQ ID NO: 43), porcine ligand peptide (1-34) (SEQ ID NO: 11) Alternatively, 1 μl of rat galanin (manufactured by Peptide Research Laboratories) (SEQ ID NO: 61) was added, and the mixture was added at 25 ° C.
For 60 minutes. Add 1.5 ml of 0.0
5% 3-[(3-cholamidopropyl) dimethylmonio] propanesulfonic acid (CHAPS), 0.1
% BSA, 5 mM MgCl ₂ , 1 mM EDTA, 50 mM Tris buffer (pH 7.4) was added, and GF was added.
/ F glass fiber filter paper (manufactured by Whatman). After the filter paper was washed with 1.5 ml of the same buffer, the radioactivity was measured with a gamma counter. 100 μM
The binding amount obtained when 1 μl of rat galanin was added was defined as the non-specific binding amount (NSB), and the binding amount obtained when 1 μl of dimethyl sulfoxide was added was defined as the maximum binding amount (B ₀ ). The specific binding amount (B ₀ -NSB) is 5
The concentration IC _{50 of} the peptide that inhibited to 0% was determined (Table 4).

[Table 4]

Reference Example 18 Cloning of cDNA Encoding Rat-Type Ligand Peptide (1-60) TRIZ was prepared from the rat hypothalamus in the same manner as in Reference Example 5.
Total RNA was prepared using OL reagent (Gibco BRL). An oligo dT cellulose column (m
RNA Purification Kit, Pharmacia)
(A) ⁺ RNA was purified. ZAP-cDNA Gi from the poly (A) ⁺ RNA
A rat hypothalamus cDNA phage library was prepared using gapack III Gold cloning kit (Stratagene). The obtained phage (2,200,000 plaque formi
ng units) of E. coli XL1-Blue MRF '(Stratagene)
Was spread on 130 NZY medium agar plates and cultured at 37 ° C. for 8 hours. The phage was transferred from the obtained E. coli plaque to Hybond N ⁺ nylon membrane (Amersham). The nylon membrane was sequentially immersed in a 0.5 N sodium hydroxide solution containing 1.5 M sodium chloride, a 0.5 M tris buffer solution (pH 7.0) containing 1.5 M sodium chloride, and a 2 × SSC solution, and then air-dried. I let it. The obtained nylon membrane was mixed with a hybridization buffer (5x SSPE, 5xDenhardts).
Solution, 0.1% SDS, 0.1 mg / ml salmon sperm DNA)
° C. To incubated for 24 hours and then hybridization buffer containing hybridization probe ^{(2.8 × 10 6 cpm / ml} ) at in (Id), in 60 ° C. 18
Incubated for hours. The hybridization probe was an EcoRI / BglII digested fragment of plasmid pGR2PL6 (SEQ ID NO: 45).
Using as a template, the following primers pGAL1-1F, pGAL88-1R
Was used (365 base pairs). ExTaq (Takara Shuzo Co., Ltd.)
0.5 μl of the attached 10x PCR buffer (500 mM KCl-25
mM MgCl ₂ -100 mM Tris · HCl, pH 8.3)
8 μl of the mM dNTP mixture, [α- ³² P] dCTP (600
(0 Ci / mmol), 1 μl each of the primers pGAL9-3F and pGAL34-8R (10 μM each), 1 μl of the template cDNA, and 54 μl of distilled water. After denaturation, 94
A cycle reaction of 20 seconds at 60 ° C. for 30 seconds to 30 seconds at 72 ° C. for 30 seconds was performed 32 times, and a final extension reaction at 72 ° C. for 4 minutes was performed. pGAL1-1F: 5'-ATGGCTCTGACTGTCCCTCTGATCGTTCT-3 '(SEQ ID NO: 46) pGAL88-1R: 5'-TGAAACCTCGTAGTTCCTGGTCGGATTCG-3' (SEQ ID NO: 47) Nylon membrane after hybridization contains 0.1% SDS2 × 5 using SSC buffer
After washing at 0 ° C, X-ray film (Kodak, BioMax
MS) in the presence of an intensifying screen (exposure conditions: -70 ° C, 3 days). Positive plaques revealed from the results of autoradiography were extracted, and S containing 0.1 ml of chloroform was added.
M buffer (100 mM NaCl, 8 mM MgSO ₄ , 0.01% gelatin,
Phage was extracted into 5 ml of 50 mM Tris-HCl, pH 7.5. The obtained phage was again infected into Escherichia coli XL1-Blue MRF 'and spread on an NZY medium agar plate.
Cultured for hours. The phage was copied from the obtained Escherichia coli plaque onto a nylon membrane (Amersham, Hybond N ⁺ ) and hybridized in the same manner as described above. A single positive clone was selected by autoradiography and extracted with a capillary. The same series of operations was repeated once again to obtain a completely single phage clone. Next, a plasmid was isolated from the cloned phage by the following method. The phage solution (1 μl) was added to E. coli XPORT (50
μl), helper phage (10 μl), E. coli XLO
The mixture was mixed with LR (5 ml), spread on an NZY agar plate, and cultured at 37 ° C. for 8 hours. A small colony of Escherichia coli XLOLR generated in the Escherichia coli XPORT plaque was picked up with a toothpick and cultured overnight at 37 ° C. on an LB medium agar plate containing ampicillin and tetracycline. Escherichia coli XLOLR was picked up from a single colony and liquid-cultured in an LB medium. After completion of the culture, E. coli XLOLR was collected, and the plasmid was purified using a plasmid purification kit (Qiagen, QIAgen 8 Well Ultra). Dye terminator Cycle Seque
ncing Kit (Applied Biosystems, PerkinElmer), followed by sequencing reaction with DNA sequencer Prism 377 (Applied Biosystems, PerkinElmer).
(ed Biosystems, PerkinElmer). As a result, rat-type ligand full-length peptide (1-6
974 bp DNA fragment (SEQ ID NO: 0)
39) could be obtained. Escherichia coli transformed with the plasmid was used for Escherichi
a coli TOP10 / pGR2RL4.

Reference Example 19 Human-type ligand peptide (1
-60) Cloning from human brain poly (A) ⁺ RNA (CLONTECH), ZAP-cDNA Synth
A human brain cDNA library was prepared using esis Kit (Stratagene). PCR was performed using the obtained cDNA as a template and primers F / R120 and R / R120. F / R120: 5'-AGGCTGGACCCTCAATAGTGCTGGTTAC-3 '(SEQ ID NO: 48) R / R120:
5'-CCATCTATGGCCTTCCACAGGTCTAGGA-3 '(SEQ ID NO: 49) The composition of the PCR reaction solution was Taq (Takara Shuzo Co., Ltd.) of 0.5.
μl, attached 10x PCR buffer (500 mM KCl-25 mM MgCl _2-
5 μl of 100 mM Tris · HCl, pH 8.3), 2.5 mM dNTP mi
xture, 4 μl of 25 mM MgCl ₂ , 0.25 ml of each of primers F / R120 and R / R120 (100 μM each), 1 μl of template cDNA, and 36 μl of distilled water were prepared, and the reaction conditions were 94 ° C.・ After 30 seconds of initial denaturation, 94 ℃ ・ 30sec-60 ℃ ・ 30sec-72 ℃
35 cycles of 60 second cycles and a final extension reaction of 10 minutes at 72 ° C. TOPO TA
Cloning was performed using a Cloning Kit (Invitrogen) according to the method described in the attached manual. The cloned DNA sequence was decoded by the method described in Reference Example 5 to obtain the sequence of SEQ ID NO: 50. AGGCTGGACC CTCAATAGTG CTGGTTACCT TCTGGGTCCC GTCCTCCACC TTCCCCAAAT 60 GGGTGACCAA GACGGAAAGA GGGAGACAGC CCTTGAGATC CTAGACCTGT GGAAGGCCAT 120 AGATGG 126 (SEQ ID NO: 50) Primer 1F / H120 from the obtained sequence
(SEQ ID NO: 51) and 1R / H120 (SEQ ID NO: 52) were prepared and used in the 5′RACE and 3′RACE experiments described below. 1F / H120: 5'-CAAATGGGTGACCAAGACGGAAAGAGGG-3 '(SEQ ID NO: 51) 1R / H120: 5'-GGTCTAGGATCTCAAGGGCTGTCTCCCT-3' (SEQ ID NO: 52) PCR reaction solution of 5'RACE and 3'RACE was Taq (Takara Shuzo Co., Ltd.) 0.5 μl and the attached 10x PCR buffer (500 mM K
5 μl of Cl-25 mM MgCl ₂ -100 mM Tris-HCl, pH 8.3)
1, 2.5 mM dNTP mixture 4 μl, 25 mM MgCl
₂ 3 μl, 10 μM Primer F / R120 (for 3′RACE) or 10 μM Primer R / R120 (for 5′RACE) 1 ml, 10 μM Primer AP1 (primer AP
1 is 1 μl of CLONTECH's Marathon-Ready cDNA Kit and a template cDNA (CLONTECH, Marathon-R)
5 μl of eady cDNA Kit, Human Hypothalamus) and 31 μl of distilled water. Reaction conditions are 94
After initial denaturation at 60 ° C for 60 seconds, 94 ° C for 30 seconds-72 ° C at 12
5 cycles of 0 second cycle, 94 ° C for 30 seconds -70 ° C for 1
The cycle reaction of 20 seconds was performed 5 times at 94 ° C for 30 seconds at -68 ° C
The 120 second cycle reaction was 25 times and a final extension reaction at 68 ° C. for 10 minutes. Subsequently, nested PCR was performed using the reaction solution of the PCR reaction as a template. The reaction solution is Taq
(Takara Shuzo Co., Ltd.) 0.5μl, 10x PCR buff attached
er (500 mM KCl-25 mM MgCl ₂ -100 mM Tris-HCl, pH 8.
3) 5 μl, 2.5 mM dNTP mixture 4 μl, 25 mM
3 μl of MgCl ₂ , 10 μM primer 1F / H120
(For 3'RACE) or 10μM Primer 1R / H120
1 μl (for 5′RACE), 10 μM primer AP2
(Primer AP2 is CLONTECH's Marathon-Ready cDNA K
1 μl), 5 μl of template DNA (50-fold diluted solution of the PCR reaction solution), and 31 μl of distilled water were prepared. The reaction conditions were as follows: after initial denaturation at 94 ° C for 60 seconds, five cycles of 94 ° C for 30 seconds to 72 ° C for 120 seconds, five cycles of 94 ° C for 30 seconds to 70 ° C for 120 seconds, and 94 cycles. A cycle reaction of 30 seconds at 68 ° C. for 120 seconds was performed 25 times and a final extension reaction at 10 minutes at 68 ° C. The obtained DNA fragment was used in TOPO TA Cloning Kit (Invit
(Rogen) according to the method described in the attached manual. The cloned DNA sequence was deciphered by the method described in Reference Example 5 (Section of porcine cDNA cloning) to obtain sequence information of the 5 'end and 3' end. Based on this sequence information, primers 1F / H470 and 1R / H450 were prepared. 1F / H470: 5'-GAGGAGCCAGAGAGAGCTGCGGAGAG-3 '(SEQ ID NO: 53) 1R / H470: 5'-GAGCTGGAGAAGAAGGATAGGAACAGGG-3' (SEQ ID NO: 54) Primers using the human brain cDNA library as a template
PCR was performed using F / H450 and R / H450. The PCR reaction solution was prepared by adding Pfu turbo DNA polymerase (Stratagene) to 0.
5 μl, attached 10x PCR buffer (500 mM KCl-25 mM MgC
l ₂ -100 mM Tris-HCl, pH 8.3) 5 μl, 2.5 mM dNTP
Mix 4 μl, 10 μM primer F / H470 and R / H
470, 2.5 ml each, 0.5 μl of template human whole brain cDNA
and 35 μl of distilled water. The reaction conditions are as follows: after initial denaturation at 94 ° C for 30 seconds,
35 cycles of 5 minutes at 5 ° C. and a final extension reaction of 10 minutes at 72 ° C. The obtained DNA fragment was subjected to pPCR-Scr
Cloning was performed using the ipt Amp SK ( ⁺ ) vector (Stratagene) according to the method described in the attached manual. The cloned DNA sequence was prepared in Reference Example 5 (porcine c
DNA cloning).
PGR2 having a 473 bp DNA fragment (SEQ ID NO: 40) encoding human full length peptide (1-60)
HL14 was obtained. Escherichia coli transformed with the plasmid was used for Escherichia coli TOP10 / pGR.
Named 2HL14.

Reference Example 20 Pig-type ligand peptide (1
Preparation of Structural Gene of (-60) (See FIG. 10) The structural gene of the (1-60) peptide of the present invention is obtained by adding an NdeI cleavage site adjacent to the structural gene upstream of the plasmid pGR2PL6 obtained in Reference Example 5, and Primer with methionine 1: 5'-AGCATATGGCTCCGGTCCACAGG-3 '(SEQ ID NO:
55 Kikkotec) and a primer 2: a 5'-CTGGATC having a stop codon and a BamHI cleavage site adjacent to the downstream.
Amplification was performed by PCR using CTCAGGAGGCCAACTGAGAC-3 ′ (SEQ ID NO: 56, manufactured by Greiner Japan). This gene was ligated to the pCRII-TOPO vector using the TOPO TA Cloning Kit (manufactured by Invitrogen), and pCRII / GAL
It was created. This plasmid was transformed into TOP10 One Shot competent cells, and X-gal (5-Bromo-4-Chloro-3-In
LB agar medium (1% peptone, 0.5%) containing 50 μg / ml ampicillin coated with dolyl-β-D-Galactose)
% Yeast extract, 0.5% sodium chloride, 2% agar), and cultured at 37 ° C. for 1 day. Transformants were selected using tetracycline resistance and β-galactosidase activity as indices. This transformant was transformed into an LB medium (1% peptone, 0.5 yeast extract, 50 μg / ml ampicillin).
0.5% sodium chloride) overnight.
The plasmid was recovered using iprepKit (Qiagen). Primer 3: 5'-CAGCCCTCGGCATCCTGGACC-3 '
Site-directed mutagenesis (Quick Change, STRATAGENE) using (SEQ ID NO: 57 Greiner Japan) and primer 4: 5'-GGTCCAGGATGCCGAGGGCTG-3 '(SEQ ID NO: 58 Greiner Japan) As a result, pCRII / GALdesBam in which the AvaI recognition site adjacent to BamHI in the pig-type ligand peptide (1-60) structural gene of the present invention was deleted was prepared. The nucleotide sequence of the porcine type ligand peptide (1-60) structural gene portion of the present invention in the plasmid was determined by a model of Applied Biosystems.
Confirmation was performed using a 377 DNA sequencer. 2 μg of pCRII / GALdesBam whose nucleotide sequence was confirmed was cut with NdeI,
The ends were blunted with T4 DNA polymerase (DNA Blunting Kit, manufactured by Takara Shuzo). Next, after digestion with EcoRV, 3% agarose gel electrophoresis was performed, and a DNA fragment of about 200 bp was extracted using a QIAquick Gel Extraction Kit (manufactured by Qiagen), and 25 μl of a TE buffer (10 mM Tris-HCl) was extracted.
(pH 8.0), 1 mM EDTA).

Reference Example 21 Pig-type ligand peptide (1
-60) Preparation of expression plasmid a) Preparation of fusion protein expression vector (see FIG. 11) Transformant Escherichia coli MM294 (DE3) /
Plasmid pTB960- carried in pTB960-11
11 After cutting 1 μg with XbaI and AvaI, 1% agarose gel electrophoresis was carried out, and a DNA fragment of about 4.4 kbp was QIAquick
Extract using Gel Extraction Kit (Qiagen),
Dissolved in 50 μl of TE buffer. hFGF mutein C
The S23 structural gene is deleted and Nd
Synthetic DNAs (5′-CTAGACATATGCCAGCATTGC-3 ′ (SEQ ID NO: 59) and 5′-TCGGGCAATGCTGGCATATGT-3 ′) designed to insert the eI cleavage site and initiation codon
(SEQ ID NO: 60) (each manufactured by Greiner Japan Co., Ltd.) (1 μg) was added to 100 μl of a phosphorylation reaction solution [50 mM Tris-HC
l (pH 7.6), 10 mM MgCl ₂ , 5 mM dithiothreitol, 0.1 mM
Spermidine, 0.1 mM EDTA, 1 mM ATP, 10 units T4 polynucleotide kinase (Nippon Gene)] at 37 ° C.
-The reaction was performed for 1 hour to phosphorylate the 5 'end. After completion of the reaction, phenol / chloroform extraction and ethanol precipitation were performed. The precipitate was dissolved in 50 μl of TE buffer, and the solution was kept at 80 ° C. for 10 minutes, and then gradually cooled at room temperature for annealing. The XbaI-AvaI DNA fragment and the annealing solution were ligated using Takara DNA Ligation Kit Ver2 (Takara Shuzo). That is, XbaI-AvaI DN
To 1 μl of the A fragment and 1 μl of the annealing solution, 3 μl of distilled water and 5 μl of the ligation solution I were added and reacted at 16 ° C. for 30 minutes. Escherichia coli JM109 competent cells (manufactured by Toyobo) were transformed with 10 μl of the ligation solution, and seeded on an LB agar medium containing 10 μg / ml tetracycline, and the resulting tetracycline-resistant colonies were selected. This transformant is cultured overnight in LB medium,
Plasmids were prepared using QIAprep8 Miniprep Kit (Qiagen). After cutting this plasmid with NdeI, 1%
Agarose gel electrophoresis was performed to confirm that a NdeI cleavage site was newly generated, and the plasmid was designated as plasmid pTBNde.
Next, 1 μg of plasmid pTBNde was digested with PstI, and T4
After blunting the ends with DNA polymerase (DNA Blunting Kit, Takara Shuzo), phenol / chloroform extraction,
Ethanol precipitation was performed. The obtained precipitate is mixed with TE buffer 1
After dissolving in 0 μl, digestion with NdeI, 3% agarose gel electrophoresis was performed, and a DNA fragment of about 470 bp was
Extract using el Extraction Kit (Qiagen),
Dissolved in 25 μl of TE buffer. Transformant Escherichia coli MM294 (DE3)
After cutting 1 μg of plasmid pTCII held in / pTCIId23-MPIF1 with BsmI and PvuII, 1% agarose gel electrophoresis was performed, and a DNA fragment of about 3.7 kbp was extracted using a QIAquick Gel Extraction Kit.
Dissolved in 5 μl of TE buffer. This DNA fragment is
After blunting the ends with T4 DNA polymerase using a Blunting Kit, ligation was performed. After the ligated solution was extracted with phenol / chloroform, ethanol precipitation was carried out and dissolved in 10 μl of TE buffer. This solution was digested with BamHI, blunt-ended with T4 DNA polymerase, further digested with NdeI, subjected to 1% agarose gel electrophoresis, and a DNA fragment of about 3.7 kbp was subjected to QIAquick
Extract using Gel Extraction Kit, 50 μl TE
Dissolved in buffer. About 470 bp of NdeI-Blunt DNA
4 μl of the fragment and 1 μl of an approximately 3.7 kbp NdeI-Blunt DNA fragment
5 μl of the ligation solution I was added to the mixture (1) and reacted at 16 ° C. for 30 minutes. Escherichia coli JM109 competent cells (manufactured by Toyobo Co., Ltd.) were transformed with 10 μl of the ligation solution, seeded on an LB agar medium containing 10 μg / ml tetracycline, and the resulting tetracycline-resistant colonies were selected. After culturing the transformant overnight in an LB medium, a plasmid was prepared using QIAprep8 Miniprep Kit to obtain a fusion protein expression vector pTFC. b) Construction of pig-type ligand peptide (1-60) expression strain (see FIG. 12) 1 μg of fusion protein expression vector pTFC was digested with BamHI, and T4 DNA polymerase (DNA Blunting Kit,
After blunting the ends with Takara Shuzo Co., Ltd., 1% agarose gel electrophoresis was carried out, and a DNA fragment of about 4.2 kbp was subjected to QIAq
Extraction was carried out using a uick Gel Extraction Kit (manufactured by Qiagen) and dissolved in 50 μl of TE buffer. This fragment and the structural gene of the polypeptide of the present invention prepared in Reference Example 20 were ligated using Takara DNA Ligation Kit Ver2 (Takara Shuzo). That is, 1 μl of a BamHI-cut blunting solution of pTFC and 4 μl of a structural gene solution of the polypeptide of the present invention were mixed, and 5 μl of a ligation I solution was added, followed by reaction at 16 ° C. for 30 minutes. Escherichia coli JM109 competent cells (manufactured by Toyobo Co., Ltd.) were transformed with 10 μl of the ligation solution, seeded on an LB agar medium containing 10 μg / ml tetracycline, cultured at 37 ° C. for 1 day, and the resulting tetracycline-resistant colonies were isolated. Selected. This transformant was cultured overnight in LB medium, and
The plasmid was recovered using p8 Miniprep Kit (manufactured by Qiagen), and the polypeptide expression plasmid pTFCGA of the present invention was recovered.
L. The nucleotide sequence of the structural gene portion of the fusion protein of this plasmid was confirmed using an Applied Biosystems model 377 DNA sequencer. Escherichia coli MM294 (DE3) was transformed with the expression plasmid pTFCGAL, seeded on an LB agar medium containing 10 μg / ml of tetracycline, cultured at 37 ° C. for 1 day, and a tetracycline-resistant transformant was selected. A polypeptide expression strain MM294 (DE3) / pTFCGAL was obtained.

Reference Example 22 Pig-type ligand peptide (1
-60) Culture of expression strain The polypeptide expression strain MM294 (DE) of the present invention obtained in Reference Example 21
3) L containing 5 mg / ml tetracycline with / pTFCGAL
The cells were cultured with shaking in 1 L of B medium at 37 ° C. for 16 hours. The obtained culture solution was added to 20 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, 0.0005% thiamine hydrochloride, 1.5% % Glucose, 1.0
50% casamino acid, 1.0% yeast extract)
Transfer to L-capacity fermenter, 37 ° C, aeration rate 20L / mi
n, aeration and stirring culture was performed at a stirring rotation speed of 200 rpm.
When the turbidity of the culture reached about 1200 klet units, isopropyl-β-D-thiogalactopyranoside (IPTG) was added to a final concentration of 23.8 mg / l. 0.7 minutes after 30 minutes and 150 minutes after IPTG addition
5% glucose was added, and culture was performed until 10 hours after the start of culture. The culture was centrifuged at 5000 rpm for 30 minutes to obtain about 830 g of bacterial cells.

Reference Example 23 Pig-type ligand peptide (1
Purification of -60) 600 ml of 150 m was added to 200 g of the cells obtained in Reference Example 22.
M sodium chloride, 0.1 mM p-amidinophenylmethanesulfonyl fluoride hydrochloride (APMSF),
50 mM phosphate buffer containing 0.1 mM EDTA (p
H7.0), suspend, and sonicate (BRANSON SONIFI)
ER MODEL450) and centrifugation (10000 rpm)
10 minutes), and the supernatant was discarded to obtain an inclusion body. This inclusion body was suspended in 60 ml of distilled water, and 140 ml of formic acid was added to dissolve the peptide. The peptide at the C-terminal side of methionine linking the fusion protein CS23 with the porcine ligand peptide (1-60) of the present invention In order to perform a cleavage reaction at the bond, 2 g of cyanogen bromide was added, and the mixture was treated at room temperature for 24 hours. After completion of the reaction, the solution was dialyzed overnight against distilled water, and centrifuged (10,000 rpm for 10 minutes). 50 mM supernatant
After dialysis again overnight against Tris-HCl (pH 7.5), the solution was adjusted to pH 6.
Adjust to 0 and centrifuge (10,000 rpm for 10 minutes)
Then, about 400 ml of the centrifuged supernatant was obtained. This centrifuged supernatant was CM-TOYOPEARL 650M (3.0 cm ID × 10 cm) equilibrated with 50 mM sodium acetate buffer (pH 6.0).
L, manufactured by Tosoh Corporation) at 10 ml / min, and well washed with the buffer used for equilibration.
Elution was performed with a step gradient (B = 50 mM sodium acetate buffer + 1 M sodium chloride, pH 6.0) to obtain a fraction containing the porcine ligand peptide (1-60) of the present invention. One-third of this fraction was C4P-50 (2.15 cmID × 30) equilibrated with 0.1% trifluoroacetic acid.
cmL, manufactured by Showa Denko KK at 5 ml / min, and then 33-43% B (B = 80% acetonitrile / 0.1%).
Elution was performed with a step gradient of 1% trifluoroacetic acid). The same operation is performed for the remaining 2/3 amount, and the fraction containing the porcine ligand peptide (1-60) of the present invention is collected, freeze-dried, and porcine ligand peptide (1-60) of the present invention. 67 mg of a purified product were obtained. a) Analysis using SDS-polyacrylamide gel electrophoresis The purified porcine ligand peptide (1-60) of the present invention was purified from 2% SDS, 10% glycerol, 5% 2-mercaptoethanol, 0.001% bromophenol blue. 0.0625 Tris-HCl (pH6.8) containing [Laemmli, UK: Nat
ure, 227, 680 (1979)], boiled for 3 minutes, and electrophoresed on Multigel 15/25 (Daiichi Pure Chemicals). Run the gel after electrophoresis on Rapid CBB KANTO (Kanto Chemical)
, A single band (see FIG. 13). b) Amino acid composition analysis The purified product of the porcine ligand peptide (1-60) of the present invention was subjected to gas phase hydrolysis with 6N hydrochloric acid containing 1% phenol at 110 ° C. for 24 and 48 hours to obtain an amino acid analyzer (Hitachi L-
The amino acid composition was determined using an 8500A Amino Acid Analyzer). As a result, as shown in Table 5, the results agreed with the amino acid composition predicted from the cDNA base sequence.

[Table 5] c) N-terminal amino acid sequence analysis The N-terminal amino acid sequence was determined using a gas phase protein sequencer (Applied Biosystems model 477A). As a result, as shown in Table 6, the results agreed with the amino acid composition predicted from the cDNA base sequence.

[Table 6] d) C-terminal amino acid analysis The purified porcine ligand peptide (1-60) of the present invention was subjected to gas-phase hydrolysis with anhydrous hydrazine at 100 ° C for 3.5 hours to obtain an amino acid analyzer (Hitachi L-8500A Amino Acid Analyte).
C-terminus was determined using zer). As a result, as shown in Table 7, the amino acids coincided with the amino acids predicted from the cDNA base sequence.

[Table 7]

Reference Example 24 Feeding Test Male Wistar rats (8 W) under pentobarbital anesthesia
A guide cannula was inserted into the third ventricle (AP: -7.1, L: 0.0, H: 2.0 mm). After recovery for one week after the operation, a feeding experiment was performed. Light-dark cycle 12 hours (light: 8:00 to 20:00)
And the feeding experiment started at 16:00. A microinjection cannula was attached to the rat under no anesthesia and without restraint, and the pig-type peptide (1-
60) (SEQ ID NO: 31) or 2.5 μl of PBS alone
The administration was performed at 1 / min for 4 minutes. One minute after the end of the administration, the microinjection cannula was removed, and the animals were allowed to freely ingest the standard feed. The food consumed within 90 minutes from the end of the administration was weighed to determine the amount of food consumed (FIG. 14).

Reference Example 25 Mouse-type ligand peptide c
DNA cloning Mouse testis Marathon-Ready cDNA (derived from BALB / c mouse, C
Primers F / R120 and R / R120 using LONTECH as template
(Described in WO 99/48920) to perform PCR. F / R120: 5′-AGGCTGGACCCTCAATAGTGCTGGTTAC-3 ′ (SEQ ID NO: 65) R / R120: 5′-CCATCTATGGCCTTCCACAGGTCTAGGA-3 ′ (SEQ ID NO: 66) The composition of the PCR reaction solution was 0.5 μl of ExTaq (Takara Shuzo), 5 μl of the attached 10xPCR buffer, 2.5 mM dN
4 μl of TP mixture, primers F / R120 and R / R120
(10 μM each), 0.5 μl each, and template cD
The reaction conditions were as follows: 1 μl of NA and 34.5 μl of distilled water were mixed, and the reaction conditions were as follows: initial denaturation at 94 ° C. for 30 seconds;
A cycle reaction of 0 second-62 ° C./30 seconds-72 ° C./60 seconds was performed 35 times, and a final extension reaction was performed at 72 ° C. for 10 minutes.
The obtained DNA fragment is subjected to TOPO TA Cloning Kit (Invitroge
n company) according to the method described in the attached manual. The sequence of the cloned DNA was decoded using ABI 377 DNA sequencer, and the sequence of SEQ ID NO: 67 was obtained. AGGCTGGACC CTCAATAGTG CTGGTTACCT CCTGGGTCCT GTCCTCCCCG TTTCCTCCAA 60 GGCCGACCAG GGCAGGAAGA GAGACTCAGC TCTTGAGATC CTAGACCTGT GGAAGGCCAT 120 AGATGG 126 (SEQ ID NO: 67) Primer 1F / M120 from the obtained sequence
(SEQ ID NO: 68) and 1R / M120 (SEQ ID NO: 69) were prepared and used in the 5 ′ RACE and 3 ′ RACE experiments described below. 1F / M120: 5'-TCCAAGGCCGACCAGGGCAGGAAGAGAG-3 '(SEQ ID NO: 68) 1R / M120: 5'-GGTCTAGGATCTCAAGAGCTGAGTCTCT-3' (SEQ ID NO: 69) PCR reaction solution for 5'RACE and 3'RACE was Taq (Takara Shuzo Co., Ltd.) 0.5 μl and the attached 10x PCR buffer (500 mM K
5 μl of Cl-25 mM MgCl ₂ -100 mM Tris-HCl, pH 8.3)
1, 2.5 mM dNTP mixture 4 μl, 25 mM MgCl
₂ 3 μl, 10 μM primer F / R120 (for 3′RACE) or 10 μM primer R / R120 (for 5′RACE) 1 μl, 10 μM primer AP1 (primer AP1)
1 is 1 µl of CLONTECH's Marathon-Ready cDNA Kit and the template cDNA (CLONTECH, mouse testis)
5 μl of Marathon-Ready cDNA) and 31 μl of distilled water
1 was mixed. The reaction conditions were as follows: after initial denaturation at 94 ° C for 60 seconds, five cycles of 94 ° C for 30 seconds to 72 ° C for 120 seconds, five cycles of 94 ° C for 30 seconds to 70 ° C for 120 seconds, and 94 cycles. A cycle reaction of 30 seconds at 68 ° C. for 120 seconds was performed 25 times and a final extension reaction at 10 minutes at 68 ° C. was performed. Subsequently, the reaction solution of the PCR reaction is used as a template for ne
Sted PCR was performed. The reaction solution was 0.5 µl of Taq (Takara Shuzo Co., Ltd.) and the attached 10x PCR buffer (500 mM KCl
5 μl of -25 mM MgCl ₂ -100 mM Tris · HCl, pH 8.3)
4 μl of 2.5 mM dNTP mixture, 3 μl of 25 mM MgCl ₂ , 10 μM Primer 1F / M120 (for 3'RACE)
Alternatively, 10 μM primer 1R / M120 (for 5'RACE)
1 μl, 10 μM primer AP2 (primer AP2 is CL
1 µl of the template DNA (attached to ONTECH's Marathon-Ready cDNA Kit), 5 µl of template DNA (50-fold dilution of the PCR reaction solution), and 31 µl of distilled water were prepared. The reaction conditions are as follows: after initial denaturation at 94 ° C for 60 seconds,
A cycle reaction of 72 ° C. for 120 seconds was performed 5 times, and a cycle reaction of 94 ° C. for 30 seconds to 70 ° C. for 120 seconds was performed 5 times, and a cycle reaction of 94 ° C.
25 cycles of -68 ° C for 120 seconds and 6
A final extension reaction was performed at 8 ° 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. Decoding the cloned DNA sequence, 5 'end,
Sequence information of the 3 'end was obtained. From this sequence information, primer 1F
/ M650, 1R / M650 were created. 1F / H650: 5'-GAAAGACAGTCTCAAATCAAGGACCTGC-3 '(SEQ ID NO: 70) 1R / H650: 5'-TGAGCTGGAAACGAAGGAGGAGTGAGGT-3' (SEQ ID NO: 71) Balb / c mouse-derived testis poly (A) + RNA (Nippon Gene) Supersrcipt II reverse transcrip
Single strand DNA was synthesized using tase (GIBCO / BRL). Using this DNA as a template, primers F / M650 and R / M6
PCR was performed using 50. PCR reaction solution is Pfu DNA
1 μl of polymerase (Stratagene), 10x PCR attached
5 μl of buffer, 4 μl of 2.5 mM dNTP mixture, 10 μl
2.5 μl of each of μM primers F / M650 and R / M650, 1 μl of template DNA, and 34.5 μl of distilled water were prepared by mixing. The reaction conditions were as follows: initial denaturation at 94 ° C for 30 seconds.
A cycle reaction of 94 ° C./30 seconds-67 ° C./30 seconds-72 ° C./4 minutes was performed 30 times, and a final extension reaction was performed at 72 ° C./10 minutes. The obtained DNA fragment was used for pCRII Blunt TOPO vecto
Using r (Invitrogen), cloning was performed according to the method described in the attached manual. The cloned DNA sequence was sequenced by a conventional method, and 180b encoding a mouse GALR2 ligand full-length peptide was synthesized.
pGR2ML1 having a DNA fragment of p (SEQ ID NO: 64) was obtained. Escherichia coli TOP10 transformed with the plasmid was named TOP10 / pGR2ML1. The amino acid sequence deduced from the base sequence of 180 base pairs is shown in SEQ ID NO: 63. The mature part is shown in SEQ ID NO: 62. Amino acid sequence of the mature portion,
Comparison with the amino acid sequences of porcine small intestinal galanin (galanin), porcine GalR2 ligand (P-GALR2L), rat GalR2 ligand (R-GALR2L) and human GalR2 ligand (H-GALR2L) revealed that the amino acid sequence of the matured portion Showed high homology to porcine GalR2 ligand (P-GALR2L), rat GalR2 ligand (R-GALR2L) and human GalR2 ligand (H-GALR2L) (see FIG. 18).

Example 1 The effect of the third intraventricular administration of GALP on pituitary hormone concentrations in plasma was examined. Adult male Wistar rats (body weight 350-380 g at the time of surgery) were anesthetized by intraperitoneal administration of pentobarbital 50 mg / kg, and fixed to a rat stereotaxic apparatus. The incisor bar was lowered 3.3 mm from the interoral line. The skull was exposed and a hole was drilled in the third ventricle using a dental drill to implant a guide cannula AG-8 (inner diameter 0.4 mm, outer diameter 0.5 mm, Acom). In addition, anchor screws were buried around the four locations. Stainless steel guide cannula, A
G-12 was inserted such that its tip was located above the third ventricle. According to the atlas of Paxinos and Watson (1998), the localization coordinates are AP: -0.8 mm from Bregma,
L: 0.0 mm, H: -0.54 mm. The guide cannula was fixed to the skull with flash glue, dental cement and anchor screws. The guide cannula has a stainless steel dummy cannula, AD-12 (0.35m outside diameter)
m, Acom) and fixed with Capnite (Acom). After the operation, the rats were housed in individual cages. After the guide cannula was implanted, the rats were bred for about one week to wait for postoperative recovery, the cap night and the dummy cannula attached to the rat skull were removed, and a Teflon (registered trademark) tube (length 50 cm, inner diameter 0.1 mm) was used instead.
mm, an outer diameter of 0.35 mm, and a stainless steel microinjection cannula (0.17 mm inner diameter, 0.35 mm outer diameter, Acom) connected to an Acom. The length of the microinjection cannula is
It was adjusted so that the tip 1 mm was exposed from the guide cannula. Connect one end of a Teflon tube to a microsyringe pump and add phosphate buffered saline (pH
7.2) or rat GALP (peptide consisting of the amino acid sequence represented by SEQ ID NO: 33; 0.5, 5 nmo)
A total of 10 μl of phosphate buffered saline in which 1) was dissolved was injected into the lateral ventricle at a flow rate of 5 μl / min. After 5 minutes from the end of the injection, the microinjection cannula was removed, and the dummy cannula was fixed again with cap night. Ten minutes after the end of the injection, the head was decapitated and blood was collected. Blood was previously stored in a 50 ml tube (Corning) at 300 KIU / m containing 3 mg / ml EDTA to prevent coagulation.
300 μl of the laprotinin solution was stored. The collected blood was centrifuged (2,500 rpm, 25 minutes) using a high-speed cooling centrifuge (HIMAC 5DL, Hitachi, Ltd.), and the supernatant (plasma) was collected. Growth hormone (G
H), thyroid stimulating hormone (TSH), follicle stimulating hormone (FSH), LH, prolactin and adrenocorticotropic hormone (ACTH) by radioimmunoassay (GH,
Amersham TSH, FSH, LH and prolactin
Corporation, ACTH; Mitsubishi Kagaku). As shown in FIG. 15, the rat GALP-administered group showed a significant increase in the blood LH concentration only in a concentration-dependent manner as compared with the control group.

Example 2 Adult Wistar male rats treated in the same manner as in Example 1 above were bred for about one week after embedding the guide cannula, waited for post-operative recovery, and performed an operation for free-time blood sampling. Was. Rats that underwent the above surgery were pentobarbital 50
The animals were anesthetized by intraperitoneal administration of mg / kg. It was fixed in a dorsal position on a dissection pad, exposing the left jugular vein. Polyethylene tube SP35 (inner diameter 0.5 mm, outer diameter 0.9
mm, Natsume Seisakusho) is cut into a length of about 30 cm,
After filling with a unit / ml of heparin-containing physiological saline, it was inserted into the jugular vein by about 4.5 cm and fixed. The other end of the tube was exposed from the neck (dorsal side) through the dorsal subcutaneous area.
After waiting overnight after surgery, rat GALP (SEQ ID NO: 33)
Prior to the administration of the peptide having the amino acid sequence represented by the formula (1), 300 µl of blood was collected using a 1 ml tuberculin syringe and a 25 gauge injection needle (both from Terumo). In order to prevent blood coagulation, 3 mg
300 KIU / ml aprotinin solution containing 1 / ml EDTA
0 μl was stored. The cap knight and dummy cannula attached to the skull of the rat were removed, and a stainless steel microinjection cannula (0.17 m inner diameter) connected to a Teflon tube (50 cm long, 0.1 mm inner diameter, 0.35 mm outer diameter, Acom) was used instead.
m, outer diameter 0.35 mm, Acom). The length of the microinjection cannula is
The mm was adjusted to be exposed from the guide cannula. One end of a Teflon tube is connected to a micro syringe pump, and 5 μl of phosphate buffered saline (pH 7.2), rat GALP (0.5, 5 nmol) or phosphate buffered saline in which rat galanin (5 nmol) is dissolved A total of 10 μl was injected into the third ventricle at a flow rate of / min. After 5 minutes from the end of the injection, the microinjection cannula was removed, and the dummy cannula was fixed again with cap night. 0 from the start of intraventricular administration,
Blood was collected from the jugular vein 300 μl each at 10, 20, 30, and 60 minutes. The collected blood is a micro high-speed refrigerated centrifuge (MR-
Centrifugation (13,000 rpm, 150, Tommy Seiko)
5 minutes), and the supernatant (plasma) was collected. LH contained in plasma was measured using a radioimmunoassay (Amersham). As shown in FIG. 16, the GALP administration group showed a tendency to increase in the GALP 1 nmol administration group compared to the control group, and the GALP 5 nmol administration group showed a significant blood LH at any blood sampling time with a peak at 30 minutes after administration.
There was an increase in concentration. On the other hand, no change in blood LH concentration was observed in the galanin administration group.

Example 3 Effect of third ventricular administration of GALP on LH concentration in plasma Zucker obese with abnormal leptin receptor
The study was conducted using rats. Adult male Zucker obese rat (weight at surgery: fatty; 410-430 g, lean; 290-310 g)
Was anesthetized by intraperitoneal administration of 50 mg / kg of pentobarbital, and fixed to a rat stereotaxic apparatus. The incisor bar was lowered 3.3 mm from the interoral line. The skull was exposed and a hole was drilled in the third ventricle using a dental drill to implant a guide cannula AG-8 (inner diameter 0.4 mm, outer diameter 0.5 mm, Acom). In addition, anchor screws were buried around the four locations. A stainless steel guide cannula, AG-12, was inserted such that its tip was located above the third ventricle. The localization coordinates are Paxinos
According to the atlas of Watson and Watson (1998), Zucker fatty acid, from bregma, AP: -1.0 mm, L: 0.0 mm,
H: 5.6mm, Zucker lean, A from Bregma
P: -0.9 mm, L: 0.0 mm, H: 5.4 mm,
And The guide cannula was fixed to the skull with flash glue, dental cement and anchor screws. Stainless steel dummy cannula, AD-12 for guide cannula
(Outer diameter 0.35 mm, Acom) was inserted and fixed with Capnite (Acom). After the operation, the rats were housed in individual cages. After the guide cannula was implanted, the animals were bred for about one week and waited for post-operative recovery, and surgery for free-time blood sampling was performed as in the example. Wait one night after surgery,
Prior to the administration of GALP, a 300 ml injection tube for tuberculin and a 25-gauge injection needle (both from Terumo) were used to prepare 300 ml.
μl of blood was collected. In order to prevent blood coagulation, the syringe should contain 300 KIU / ml apap containing 3 mg / ml EDTA in advance.
10 μl of the rotinin solution was stored. The cap knight and the dummy cannula attached to the rat skull were removed, and a stainless steel microinjection cannula (0.17 mm ID) connected to a Teflon tube (50 cm long, 0.1 mm ID, 0.35 mm outer diameter, Acom) was used instead. , 0.35mm outside diameter, Acom)
Was inserted. The length of the microinjection cannula was adjusted so that the tip 1 mm was exposed from the guide cannula. One end of the Teflon tube was connected to a micro syringe pump, and phosphate buffered saline (pH 7.2) and phosphate buffered saline in which rat GALP (1 nmol) was dissolved were added at a flow rate of 5 μl / min for a total of 1
0 μl was injected into the third ventricle. After 5 minutes from the end of the injection, the microinjection cannula was removed, and the dummy cannula was fixed again with cap night. At 0, 10, 20, 30, and 60 minutes after the start of intracerebroventricular administration, 300 μl of blood was collected from the jugular vein. The collected blood is centrifuged (13,000 rpm, 5 minutes) using a micro high-speed cooling centrifuge (MR-150, Tommy Seiko), and the supernatant (plasma)
Was recovered. LH contained in plasma was measured using a radioimmunoassay (Amersham). As shown in FIG. 17, in Zucker fatty acid, the GALP administration group had significant blood LH levels at 10, 20, and 30 minutes after administration compared to the control group.
There was an increase in concentration. On the other hand, in Zucker lean, GA
No significant increase in blood LH concentration was observed in the LP administration group as compared to the control group, and the blood LH increasing effect of GALP was enhanced in Zucker fatty as compared to Zucker lean.

[0077]

EFFECT OF THE INVENTION Since the polypeptide of the present invention has an LH secretion promoting action, it can be used as an agent for preventing and treating various diseases related to LH secretion deficiency. On the other hand, the peptide of the present invention has a high affinity for its receptor protein, so that when the dose is increased, desensitization of LH secretion occurs, and as a result, the peptide also has an effect of suppressing LH secretion. In this case, it can be used as an agent for preventing or treating various diseases related to LH hypersecretion.

[0078]

[Sequence listing] [Sequence Listing] <110> Takeda Chemical Industries, Ltd. <120> Use of Galanin-like peptide <130> P01-0291 <160> 72 <210> 1 <211> 346 <212> PRT <213 > Rat <400> 1 Met Glu Leu Ala Pro Val Asn Leu Ser Glu Gly Asn Gly Ser Asp Pro 1 5 10 15 Glu Pro Pro Ala Glu Pro Arg Pro Leu Phe Gly Ile Gly Val Glu Asn 20 25 30 Phe Ile Thr Leu Val Val Phe Gly Leu Ile Phe Ala Met Gly Val Leu 35 40 45 Gly Asn Ser Leu Val Ile Thr Val Leu Ala Arg Ser Lys Pro Gly Lys 50 55 60 Pro Arg Ser Thr Thr Asn Leu Phe Ile Leu Asn Leu Ser Ile Ala Asp 65 70 75 80 Leu Ala Tyr Leu Leu Phe Cys Ile Pro Phe Gln Ala Thr Val Tyr Ala 85 90 95 Leu Pro Thr Trp Val Leu Gly Ala Phe Ile Cys Lys Phe Ile His Tyr 100 105 110 Phe Phe Thr Val Ser Met Leu Val Ser Ile Phe Thr Leu Ala Ala Met 115 120 125 Ser Val Asp Arg Tyr Val Ala Ile Val His Ser Arg Arg Ser Ser Ser 130 135 140 Leu Arg Val Ser Arg Asn Ala Leu Leu Gly Val Gly Phe Ile Trp Ala 145 150 155 160 Leu Ser Ile Ala Met Ala Ser Pro Val Ala Tyr Tyr Gln Arg Leu Phe 165 170 175 His Arg Asp Ser Asn Gln Thr Phe Cys Trp Glu His Trp Pro Asn Gln 180 185 190 Leu His Lys Lys Ala Tyr Val Val Cys Thr Phe Val Phe Gly Tyr Leu 195 200 205 Leu Pro Leu Leu Leu Ile Cys Phe Cys Tyr Ala Lys Val Leu Asn His 210 215 220 Leu His Lys Lys Leu Lys Asn Met Ser Lys Lys Ser Glu Ala Ser Lys 225 230 235 240 Lys Lys Thr Ala Gln Thr Val Leu Val Val Val Val Val Phe Gly Ile 245 250 255 Ser Trp Leu Pro His His Val Ile His Leu Trp Ala Glu Phe Gly Ala 260 265 270 Phe Pro Leu Thr Pro Ala Ser Phe Phe Phe Arg Ile Thr Ala His Cys 275 280 285 Leu Ala Tyr Ser Asn Ser Ser Val Asn Pro Ile Ile Tle Ayr Phe Leu 290 295 300 Ser Glu Asn Phe Arg Lys Ala Tyr Lys Gln Val Phe Lys Cys Arg Val 305 310 315 320 Cys Asn Glu Ser Pro His Gly Asp Ala Lys Glu Lys Asn Arg Ile Asp 325 330 335 Thr Pro Pro Ser Ser Thr Asn Cys Thr His Val 340 345 <210> 2 <211> 372 <212> PRT <213> Rat <400> 2 Met Asn Gly Ser Gly Ser Gln Gly Ala Glu Asn Thr Ser Gln Glu Gly 1 5 10 15 Gly Ser Gly Gly Trp Gln Pro Glu Ala Val Leu Val Pro Leu Phe Phe 20 25 30 Ala Leu Ile Phe Leu Val Gly Thr Val Gly Asn Ala Leu Val Leu Ala 35 40 45 Val Leu Leu Arg Gly Gly Gln Ala Val Ser Thr Thr Asn Leu Phe Ile 50 55 60 Leu Asn Leu Gly Val Ala Asp Leu Cys Phe Ile Leu Cys Cys Val Pro 65 70 75 80 Phe Gln Ala Thr Ile Tyr Thr Leu Asp Asp Trp Val Phe Gly Ser Leu 85 90 95 Leu Cys Lys Ala Val His Phe Leu Ile Phe Leu Thr Met His Ala Ser 100 105 110 Ser Phe Thr Leu Ala Ala Val Ser Leu Asp Arg Tyr Leu Ala Ile Arg 115 120 125 Tyr Pro Leu His Ser Arg Glu Leu Arg Thr Pro Arg Asn Ala Leu Ala 130 135 140 Ala Ile Gly Leu Ile Trp Gly Leu Ala Leu Leu Phe Ser Gly Pro Tyr 145 150 155 160 Leu Ser Tyr Tyr Arg Gln Ser Gln Leu Ala Asn Leu Thr Val Cys His 165 170 175 Pro Ala Trp Ser Ala Pro Arg Arg Arg Ala Met Asp Leu Cys Thr Phe 180 185 190 Val Phe Ser Tyr Leu Leu Pro Val Leu Val Leu Ser Leu Thr Tyr Ala 195 200 205 Arg Thr Leu Arg Tyr Leu Trp Arg Thr Val Asp Pro Val Thr Ala Gly 210 215 220 Ser Gly Ser Gln Arg Ala Lys Arg Lys Val Thr Arg Met Ile Ile Ile 225 230 235 240 Val Ala Val Leu Phe Cys Leu Cys Trp Met Pro His His Ala Leu Ile 245 250 255 Leu Cys Val Trp Phe Gly Arg Phe Pro Leu Thr Arg Ala Thr Tyr Ala 260 265 270 270 Leu Arg Ile Leu Ser His Leu Val Ser Tyr Ala Asn Ser Cys Val Asn 275 280 285 Pro Ile Val Tyr Ala Leu Val Ser Lys His Phe Arg Lys Gly Phe Arg 290 295 300 Lys Ile Cys Ala Gly Leu Leu Arg Pro Ala Pro Arg Arg Ala Ser Gly 305 310 315 320 Arg Val Ser Ile Leu Ala Pro Gly Asn His Ser Gly Ser Met Leu Glu 325 330 335 Gln Glu Ser Thr Asp Leu Thr Gln Val Ser Glu Ala Ala Gly Pro Leu 340 345 350 Val Pro Pro Pro Ala Leu Pro Asn Cys Thr Ala Ser Ser Arg Thr Leu 355 360 365 Asp Pro Ala Cys 370 <210> 3 <211> 370 <212> PRT <213> Rat <400> 3 Met Ala Asp Ile Gln Asn Ile Ser Leu Asp Ser Pro Gly Ser Val Gly 1 5 10 15 Ala Val Ala Val Pro Val Ile Phe Ala Leu Ile Phe Leu Leu Gly Met 20 25 30 Val Gly Asn Gly Leu Val Leu Ala Val Leu Leu Gln Pro Gly Pro Ser 35 40 45 Ala Trp Gln Glu Pro Ser Ser Thr Thr Asp Leu Phe Ile Leu Asn Leu 50 55 60 Ala Val Ala Asp Leu Cys Phe I le Leu Cys Cys Val Pro Phe Gln Ala 65 70 75 80 Ala Ile Tyr Thr Leu Asp Ala Trp Leu Phe Gly Ala Phe Val Cys Lys 85 90 95 Thr Val His Leu Leu Ile Tyr Leu Thr Met Tyr Ala Ser Ser Phe Thr 100 105 110 Leu Ala Ala Val Ser Leu Asp Arg Tyr Leu Ala Val Arg His Pro Leu 115 120 125 Arg Ser Arg Ala Leu Arg Thr Pro Arg Asn Ala Arg Ala Ala Val Gly 130 135 140 Leu Val Trp Leu Leu Ala Ala Leu Phe Ser Ala Pro Tyr Leu Ser Tyr 145 150 155 160 Tyr Gly Thr Val Arg Tyr Gly Ala Leu Glu Leu Cys Val Pro Ala Trp 165 170 175 Glu Asp Ala Arg Arg Arg Ala Leu Asp Val Ala Thr Phe Ala Ala Gly 180 185 190 Tyr Leu Leu Pro Val Ala Val Val Ser Leu Ala Tyr Gly Arg Thr Leu 195 200 205 Cys Phe Leu Trp Ala Ala Val Gly Pro Ala Gly Ala Ala Ala Ala Glu 210 215 220 Ala Arg Arg Arg Ala Thr Gly Arg Ala Gly Arg Ala Met Leu Ala Val 225 230 235 240 Ala Ala Leu Tyr Ala Leu Cys Trp Gly Pro His His Ala Leu Ile Leu 245 250 255 Cys Phe Trp Tyr Gly Arg Phe Ala Phe Ser Pro Ala Thr Tyr Ala Cys 260 265 270 Arg Leu Ala Ser His Cys Leu Ala Tyr Ala Asn Ser Cys Leu Asn Pro 275 280 285 Leu Val Tyr Ser Leu Ala Ser Arg His Phe Arg Ala Arg Phe Arg Arg 290 295 300 Leu Trp Pro Cys Gly Arg Arg Arg His Arg His His His Arg Ala His 305 310 315 320 Arg Ala Leu Arg Arg Val Gln Pro Ala Ser Ser Gly Pro Ala Gly Tyr 325 330 335 Pro Gly Asp Ala Arg Pro Arg Gly Trp Ser Met Glu Pro Arg Gly Asp 340 345 350 Ala Leu Arg Gly Gly Gly Glu Thr Arg Leu Thr Leu Ser Pro Arg Gly 355 360 365 Pro Gln 370 <210> 4 <211> 1041 <212> DNA <213> Rat <400> 4 ATGGAACTGG CTCCGGTGAA CCTCAGTGAA GGGAATGGGA GCGACCCTGA ACCTCCAGCG 60 GAACCCAGGC CGCTCTTCGG CATCGGCGTG GAGAACTTCA TCACGCTGGT GGTGTTTGGC 120 CTTATTTTCG CGATGGGCGT GCTGGGCAAC AGCCTGGTGA TCACCGTGCT GGCGCGCAGC 180 AAACCGGGCA AGCCGCGCAG CACCACCAAC CTGTTCATCC TCAACCTGAG CATCGCAGAC 240 CTGGCCTACC TGCTCTTCTG CATCCCTTTC CAGGCCACCG TGTACGCACT GCCCACCTGG 300 GTGCTGGGCG CCTTCATCTG CAAGTTTATA CACTACTTCT TCACCGTGTC CATGCTCGTG 360 AGCATCTTCA CCCTGGCCGC GATGTCTGTG GATCGCTATG TGGCCATTGT GCATTCACGG 420 CGCTCCTCCT CCCTCAGGGT GTCCCG CAAC GCGCTGCTGG GCGTGGGCTT CATCTGGGCG 480 CTGTCCATCG CTATGGCCTC GCCGGTGGCC TACTACCAGC GCCTTTTTCA TCGGGACAGC 540 AACCAAACCT TCTGCTGGGA GCACTGGCCC AACCAACTCC ACAAGAAGGC TTACGTGGTG 600 TGCACTTTCG TCTTTGGTTA CCTTCTGCCC TTACTGCTCA TCTGCTTTTG CTATGCCAAG 660 GTTCTCAATC ATCTGCATAA AAAGTTGAAG AACATGTCAA AAAAGTCAGA GGCATCCAAG 720 AAAAAGACTG CACAGACTGT CCTGGTGGTC GTTGTGGTAT TTGGCATATC ATGGCTGCCC 780 CATCATGTCA TCCACCTCTG GGCTGAGTTC GGAGCATTCC CGCTGACCCC AGCTTCCTTC 840 TTCTTCAGAA TCACTGCCCA CTGCCTGGCA TACAGCAACT CCTCGGTGAA CCCCATCATC 900 TACGCCTTTC TCTCAGAAAA CTTCCGGAAG GCGTACAAGC AAGTGTTCAA GTGCCGTGTT 960 TGCAATGAGT CGCCGCACGG CGATGCTAAA GAAAAGAACC GAATAGATAC CCCGCCCTCC 1020 ACCAACTGCA CCCACGTGTG A 1041 <210> 5 <211> 1119 <212> DNA <213> Rat <400> 5 ATGAATGGCT CCGGCAGCCA GGGCGCGGAG AACACGAGCC AGGAAGGCGG TAGCGGCGGC 60 TGGCAGCCTG AGGCGGTCCT TGTACCCCTA TTTTTCGCGC TCATCTTCCT CGTGGGCACC 120 GTGGGCAACG CGCTGGTGCT GGCGGTGCTG CTGCGCGGCG GCCAGGCGGT CAGCACCACC 180 AACCTGTTCA TCCTCAACCT GGGCGTGGCC GACCTGTGT T TCATCCTGTG CTGCGTGCCT 240 TTCCAGGCCA CCATCTACAC CCTGGACGAC TGGGTGTTCG GCTCGCTGCT CTGCAAGGCT 300 GTTCATTTCC TCATCTTTCT CACTATGCAC GCCAGCAGCT TCACGCTGGC CGCCGTCTCC 360 CTGGACAGGT ATCTGGCCAT CCGCTACCCG CTGCACTCCC GAGAGTTGCG CACACCTCGA 420 AACGCGCTGG CCGCCATCGG GCTCATCTGG GGGCTAGCAC TGCTCTTCTC CGGGCCCTAC 480 CTGAGCTACT ACCGTCAGTC GCAGCTGGCC AACCTGACAG TATGCCACCC AGCATGGAGC 540 GCACCTCGAC GTCGAGCCAT GGACCTCTGC ACCTTCGTCT TTAGCTACCT GCTGCCAGTG 600 CTAGTCCTCA GTCTGACCTA TGCGCGTACC CTGCGCTACC TCTGGCGCAC AGTCGACCCG 660 GTGACTGCAG GCTCAGGTTC CCAGCGCGCC AAACGCAAGG TGACACGGAT GATCATCATC 720 GTGGCGGTGC TTTTCTGCCT CTGTTGGATG CCCCACCACG CGCTTATCCT CTGCGTGTGG 780 TTTGGTCGCT TCCCGCTCAC GCGTGCCACT TACGCGTTGC GCATCCTTTC ACACCTAGTT 840 TCCTATGCCA ACTCCTGTGT CAACCCCATC GTTTACGCTC TGGTCTCCAA GCATTTCCGT 900 AAAGGTTTCC GCAAAATCTG CGCGGGCCTG CTGCGCCCTG CCCCGAGGCG AGCTTCGGGC 960 CGAGTGAGCA TCCTGGCGCC TGGGAACCAT AGTGGCAGCA TGCTGGAACA GGAATCCACA 1020 GACCTGACAC AGGTGAGCGA GGCAGCCGGG CCCCTTGTCC CACCACCCGC ACTTCC CAAC 1080 TGCACAGCCT CGAGTAGAAC CCTGGATCCG GCTTGTTAA 1119 <210> 6 <211> 1113 <212> DNA <213> Rat <400> 6 ATGGCTGACA TCCAGAACAT TTCGCTGGAC AGCCCAGGGA GCGTAGGGGC TGTGGCAGTG 60 CCTGTGATCT TTGCCCTCAT CTTCCTGTTG GGCATGGTGG GCAATGGGCT GGTGTTGGCT 120 GTGCTACTGC AGCCTGGCCC AAGTGCCTGG CAGGAGCCAA GCAGTACCAC AGATCTCTTC 180 ATCCTCAACT TGGCCGTGGC CGACCTTTGC TTCATCCTGT GCTGCGTGCC CTTCCAGGCA 240 GCCATCTACA CACTGGATGC CTGGCTCTTT GGGGCTTTCG TGTGCAAGAC GGTACATCTG 300 CTCATCTACC TCACCATGTA TGCCAGCAGC TTCACCCTGG CGGCCGTCTC CCTGGACAGG 360 TACCTGGCTG TGCGGCACCC ACTGCGCTCC AGAGCCCTGC GCACCCCGCG CAACGCGCGC 420 GCCGCCGTGG GGCTCGTGTG GCTGCTGGCG GCTCTCTTTT CCGCGCCCTA CCTAAGCTAT 480 TACGGCACGG TGCGCTACGG CGCGCTCGAG CTCTGCGTGC CCGCTTGGGA GGACGCGCGG 540 CGGCGCGCGC TGGACGTGGC CACCTTCGCC GCGGGCTACC TGCTGCCGGT GGCCGTGGTG 600 AGCCTGGCCT ACGGACGCAC GCTATGTTTC CTATGGGCCG CCGTGGGTCC CGCGGGCGCG 660 GCGGCAGCAG AGGCGCGCAG ACGGGCGACC GGCCGGGCGG GACGCGCCAT GCTGGCAGTG 720 GCCGCGCTCT ACGCGCTTTG CTGGGGCCCG CACCACGCGCGC TCATCCTCTG C TTCTGGTAC 780 GGCCGCTTCG CCTTCAGCCC GGCCACCTAC GCCTGTCGCC TGGCCTCGCA CTGCCTCGCC 840 TACGCCAACT CCTGCCTTAA CCCGCTCGTC TACTCGCTCG CCTCGCGCCA CTTCCGCGCG 900 CGCTTCCGCC GCCTGTGGCC CTGCGGCCGT CGCCGCCACC GCCACCACCA CCGCGCTCAT 960 CGAGCCCTCC GTCGTGTCCA GCCGGCGTCT TCGGGCCCCG CCGGTTATCC CGGCGACGCC 1020 AGGCCTCGTG GTTGGAGTAT GGAGCCCAGA GGGGATGCTC TGCGTGGTGG TGGAGAGACT 1080 AGACTAACCC TGTCCCCCAG GGGACCTCAA TAA 1113 <210> 7 <211> 29 <212> PRT <213> Porcine <400> 7 Gly Trp Thr Leu Asn Ser Ala Gly Tyr Leu Leu Gly Pro His Ala Ile 1 5 10 15 Asp Asn His Arg Ser Phe His Asp Lys Tyr Gly Leu Ala 20 25 <210> 8 <211> 123 <212> PRT <213> Porcine <400> 8 Met Pro Arg Gly Cys Ala Leu Leu Leu Ala Ser Leu Leu Leu Ala Ser 1 5 10 15 Ala Leu Ser Ala Thr Leu Gly Leu Gly Ser Pro Val Lys Glu Lys Arg 20 25 30 Gly Trp Thr Leu Asn Ser Ala Gly Tyr Leu Leu Gly Pro His Ala Ile 35 40 45 Asp Asn His Arg Ser Phe His Asp Lys Tyr Gly Leu Ala Gly Lys Arg 50 55 60 Glu Leu Glu Pro Glu Asp Glu Ala Arg Pro Gly Gly Phe Asp Arg Leu 65 7 0 75 80 Gln Ser Glu Asp Lys Ala Ile Arg Thr Ile Met Glu Phe Leu Ala Phe 85 90 95 Leu His Leu Lys Glu Ala Gly Ala Leu Gly Arg Leu Pro Gly Leu Pro 100 105 110 Ser Ala Ala Ser Ser Glu Asp Ala Gly Gln Ser 115 120 <210> 9 <211> 38 <212> PRT <213> Porcine <400> 9 Leu Gly Ser Pro Val Lys Glu Lys Arg Gly Trp Thr Leu Asn Ser Ala 1 5 10 15 Gly Tyr Leu Leu Gly Pro His Ala Ile Asp Asn His Arg Ser Phe His 20 25 30 Asp Lys Tyr Gly Leu Ala 35 <210> 10 <211> 25 <212> PRT <213> Porcine <400> 10 Asn Ser Ala Gly Tyr Leu Leu Gly Pro His Ala Ile Asp Asn His Arg 1 5 10 15 Ser Phe His Asp Lys Tyr Gly Leu Ala 20 25 <210> 11 <211> 34 <212> PRT <213> Porcine <400> 11 Ala Pro Val His Arg Gly Arg Gly Gly Trp Thr Leu Asn Ser Ala Gly 1 5 10 15 Tyr Leu Leu Gly Pro Val Leu His Pro Pro ser Xaa Ala Glu Gly Gly 20 25 30 Gly Lys <210> 12 <211> 32 <212> PRT <213> Porcine <400 > 12 Ala Pro Val His Arg Gly Arg Gly Gly Trp Thr Leu Asn Ser Ala Gly 1 5 10 15 Tyr Leu Leu Gly Pro Val Leu His Pro Pro ser Xaa Ala Glu Gly Gly 20 25 30 <210> 13 <211> 9 <212> PRT <213> Porcine <400> 13 Ala Pro Val His Arg Gly Arg Gly Gly 1 5 <210> 14 <211> 6 <212> PRT <213> Porcine <400 > 14 Thr Leu Asn Ser Ala Gly 1 5 <210> 15 <211> 27 <212> PRT <213> Porcine <400> 15 Leu Leu Gly Pro Val Leu His Pro Pro Ser Xaa Ala Glu Gly Gly Gly 1 5 10 15 Lys Gly Lys Thr Ala Leu Gly Ile Leu Asp Leu 20 25 <210> 16 <211> 11 <212> PRT <213> Porcine <400> 16 Xaa Ala Ile Asp Gly Leu Pro Tyr Pro Gln Ser 1 5 10 <210> 17 <211> 44 <212> PRT <213> Porcine <400> 17 Ala Pro Val His Arg Gly Arg Gly Gly Trp Thr Leu Asn Ser Ala Gly 1 5 10 15 Tyr Leu Leu Gly Pro Val Leu His Pro Pro ser Xaa Ala Glu Gly Gly 20 25 30 Gly Lys Gly Lys Thr Ala Leu Gly Ile Leu Asp Leu 35 40 <210> 18 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> <400> 18 GTCGACATGA ATGGCTCCGG CAGCCAG 27 <210> 19 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> <400> 19 ACTAGTTTAA CAAGCCGGAT CCAGGGTTCT AC 32 <210> 20 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> <400> 20 CAY MGNGGIMGNGGIGGSTG GAC 23 <210> 21 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> <400> 21 GGHTGGACNC TNAAYAGYGC 20 <210> 22 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> <400> 22 ATICCNAGIG CNGTYTTICC YTT 23 <210> 23 <211> 98 <212> DNA <213> Porcine <400> 23 GGCTGGACTT TAAATAGTGC TGGTTACCTC CTGGGTCCCG TACTCCATCC GCCCTCCAGG 60 GCTGAAGGAG GCGGGAAGGG CAAAACAGCC CGGCAT 98 <211> 98 <212> DNA <213> Porcine <400> 24 GGTTGGACTT TGAACAGTGC TGGTTACCTC CTGGGTCCCG TACTCCATCC GCCCTCCAGG 60 GCTGAAGGAG GCGGGAAGGG CAAAACCGCC CTAGGCAT 98 <210> 25 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> > <400> 25 GGHTGGACNC TNAAYAGYGC 20 <210> 26 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> <400> 26 ATDCCBAGGG CDGTTTTGCC CTT 23 <210> 27 <211> 974 <212> DNA <213> Porcine <400> 27 TTCAGCCTCA AGCACCCATC CCTCCAGCCC TCAGATGGCT CTGACTGTCC CTCTGATCGT 60 TCTTGCAGTC CTGCTCAGCC TGATGGAGTC TCCAGCCTCT GCTCCGGTCC ACAGGGGGCG 120 AGGAGGCTGG ACCCTCAACA GTGCTGGTTA CCTCCTGGGT CC CGTACTCC ATCCGCCCTC 180 CAGGGCTGAA GGAGGCGGGA AGGGGAAGAC AGCCCTCGGG ATCCTGGACC TGTGGAAGGC 240 CATTGATGGG CTCCCCTATC CCCAGTCTCA GTTGGCCTCC AAGAGGAGTC TGGGGGAGAC 300 TTTCGCCAAA CCAGACTCTG GAGTAACATT TGTTGGAGTT CCTGACGTGG TGCCGTGGAA 360 ACGAATCCGA CCAGGAACTA CGAGGTTTCA GATCTAGGCA AGCTCTGCAA GAACGTTCCA 420 AAGGAGAAAG ATGCCTTGCC GTCATATATG CCTCCAAACT TCCGCTCCAA ACTTCCCCCC 480 CGTCTCCAGA TCCTCCTGAA ACCCTAGGTA GACACCCTCT ACTGAGACTG GGAGCCTGAA 540 AGTAAATCCC CAAATCCCAG GTAGAAAATG GGGAGCATTT GAAGAATTAT TCTCAAAAGT 600 CCCCGGACTG TGCCAGGTTT CACTGATCCC CCCCTCCCCC TTGGACTAAG TGTAAAGCGA 660 TGTAAACCAA CTCAAGAATA ATTCTGAAAC CATTCAGGAG ATCCGGAGAG GAATCGGGAA 720 ATACTCCTGC AGTGCATTTA AAGTAACTGG GTCCTATGCA ACATGAGCCA TTGGATCATA 780 CAATATTGAT ATCCCTTCTA ACACGGAGGT TCTAGGGTGT CTCAGCTGGA AAAGATTCTT 840 CAGAGTAGCA TGCTTGCCTT ACCCCATCCT TCTCACCCCA CCCCGAGCCT CCTCCAGCAG 900 AAAGGACGAG AGGCCATCTG GAGCAGAGCA GAGAGAATAA ATATTCCCTT TCAAAGAAAA 960 AAAAAAAAAA AAAA 974 <210> 28 <211> 1007 <212> DNA <213> Porcine <4 00> 28 GGAACGAGCT GGGGAGAGCT GCCGACTGCA GGCAGCCTTC TTCAGCCTCA AGCACCCATC 60 CCTCCAGCCC TCAGATGGCT CTGACTGTCC CTCTGATCGT TCTTGCAGTC CTGCTCAGCC 120 TGATGGAGTC TCCAGCCTCT GCTCCGGTCC ACAGGGGGCG AGGAGGCTGG ACCCTCAACA 180 GTGCTGGTTA CCTCCTGGGT CCCGTACTCC ATCCGCCCTC CAGGGCTGAA GGAGGCGGGA 240 AGGGGAAGAC AGCCCTCGGG ATCCTGGACC TGTGGAAGGC CATTGATGGG CTCCCCTATC 300 CCCAGTCTCA GTTGGCCTCC AAGAGGAGTC TGGGGAGACT TTCGCCAAAC CAGACTCTGG 360 AGTAACATTT GTTGGAGTTC CTGACGTGGT GCCGTGGAAA CGAATCCGAC CAGGAACTAC 420 GAGGTTTCAG ATCTAGGCAA GCTCTGCAAG AACGTTCCAA AGGAGAAAGA TGCCTTGTCG 480 TCATATATGC CTCCAAACTT CCGCTCCAAA CTTCCCCCCC GTCCCCAGAT CCTCCTGAAA 540 CCCTAGGTAG ACACCCTCTA CTGAGACTGG GAGCCTGAAA GTAAATCCCC AAATCCCAGG 600 TAGAAAATGG GGAGCATTTG AAGAATTATT CTCAAAAGTC CCCGGACTGT GCCAGGTTTC 660 ACTGATCCCC CCCCCCCCCC TCCTTGGACT AAGTGTAAAG CGATGTAAAC CAACTCAAGA 720 ATAATTCTGA AACCATTCAG GAGATCCGGA GAGGAATCGG GAAATACTCC TGCAGTGCAT 780 TTAAAGTAAC TGGGTCCTAT GCAACATGAG CCATTGGATC ATACAATATT GATATCCCTT 840 CTAACACGGA GGT TCTAGGG TGTCTCAGCT GGAAAAGATT CTTCAGAGTA GCATGCTTGC 900 CTTACCCCAT CCTTCTCACC CCACCCCGAG CCTCCTCCAG CAGAAAGGAC GAGAGGCCAT 960 CTGGAGCAGA GCAGAGAGAA TAAATATTCC CTTTCAAAGA AAAAAAA 1007 <210> 29 <211> 120 <212> LeAu Pule Ala <P> Ala Val Leu Leu Ser Leu 1 5 10 15 Met Glu Ser Pro Ala Ser Ala Pro Val His Arg Gly Arg Gly Gly Trp 20 25 30 Thr Leu Asn Ser Ala Gly Tyr Leu Leu Gly Pro Val Leu His Pro Pro 35 40 45 Ser Arg Ala Glu Gly Gly Gly Lys Gly Lys Thr Ala Leu Gly Ile Leu 50 55 60 Asp Leu Trp Lys Ala Ile Asp Gly Leu Pro Tyr Pro Gln Ser Gln Leu 65 70 75 80 Ala Ser Lys Arg Ser Leu Gly Glu Thr Phe Ala Lys Pro Asp Ser Gly 85 90 95 Val Thr Phe Val Gly Val Pro Asp Val Val Pro Trp Lys Arg Ile Arg 100 105 110 Pro Gly Thr Thr Arg Phe Gln Ile 115 120 <210> 30 <211> 96 <212> PRT <213> Porcine <400> 30 Met Ala Leu Thr Val Pro Leu Ile Val Leu Ala Val Leu Leu Ser Leu 1 5 10 15 Met Glu Ser Pro Ala Ser Ala Pro Val His Arg Gly Arg Gly Gly Trp 20 25 30 Thr Leu Asn Ser Ala Gly Tyr Leu Leu Gly Pro Val Leu His Pro Pro 35 40 45 Ser Arg Ala Glu Gly Gly Gly Lys Gly Lys Thr Ala Leu Gly Ile Leu 50 55 60 Asp Leu Trp Lys Ala Ile Asp Gly Leu Pro Tyr Pro Gln Ser Gln Leu 65 70 75 80 Ala Ser Lys Arg Ser Leu Gly Arg Leu Ser Pro Asn Gln Thr Leu Glu 85 90 95 <210> 31 <211> 60 <212> PRT <213> Porcine <400> 31 Ala Pro Val His Arg Gly Arg Gly Gly Trp Thr Leu Asn Ser Ala Gly 1 5 10 15 Tyr Leu Leu Gly Pro Val Leu His Pro Pro Ser Arg Ala Glu Gly Gly 20 25 30 Gly Lys Gly Lys Thr Ala Leu Gly Ile Leu Asp Leu Trp Lys Ala Ile 35 40 45 Asp Gly Leu Pro Tyr Pro Gln Ser Gln Leu Ala Ser 50 55 60 <210> 32 <211> 180 <212> DNA <213> Porcine <400> 32 GCTCCGGTCC ACAGGGGGCG AGGAGGCTGG ACCCTCAACA GTGCTGGTTA CCTCCTCTGTGT 60 CCCGTACTCC ATCCGCCCTC CAGGGCTGAGGAGGAGGCAG AGCCCTCGGG 120 ATCCTGGACC TGTGGAAGGC CATTGATGGG CTCCCCTATC CCCAGTCTCA GTTGGCCTCC 180 <210> 33 <211> 60 <212> PRT <213> Rat <400> 33 Ala Pro Ala His Arg Gly Arg Gly Gly Trp Thr Leu Asn Ser Ala Gly 1 5 10 15 Tyr Leu Leu Gly Pro Val Leu His Leu Ser Ser Lys Ala Asn Gln Gly 20 25 30 Arg Lys Thr Asp Ser Ala Leu Glu Ile Leu Asp Leu Trp Lys Ala Ile 35 40 45 Asp Gly Leu Pro Tyr Ser Arg Ser Pro Arg Met Thr 50 55 60 <210> 34 <211> 60 <212> PRT <213> Human <400> 34 Ala Pro Ala His Arg Gly Arg Gly Gly Trp Thr Leu Asn Ser Ala Gly 1 5 10 15 Tyr Leu Leu Gly Pro Val Leu His Leu Pro Gln Met Gly Asp Gln Asp 20 25 30 Gly Lys Arg Glu Thr Ala Leu Glu Ile Leu Asp Leu Trp Lys Ala Ile 35 40 45 Asp Gly Leu Pro Tyr Ser His Pro Pro Gln Pro Ser 50 55 60 <210> 35 < 211> 9 <212> PRT <213> Human, Rat <400> 35 Ala Pro Ala His Arg Gly Arg Gly Gly 1 5 <210> 36 <211> 21 <212> PRT <213> Human, Rat <400> 36 Ala Pro Ala His Arg Gly Arg Gly Gly Trp Thr Leu Asn Ser Ala Gly 1 5 10 15 Tyr Leu Leu Gly Pro 20 <210> 37 <211> 123 <212> PRT <213> Rat <400> 37 Met Pro Cys Phe Ser Ser Ser Arg Met Ala Cys Ser Lys His Leu Val 1 5 10 15 Leu Phe Leu Thr Ile Leu Leu Ser Leu Ala Glu Thr Pro Asp Ser Ala 20 25 30 Pro Ala His Arg Gly Arg Gly Gly Tr p Thr Leu Asn Ser Ala Gly Tyr 35 40 45 Leu Leu Gly Pro Val Leu His Leu Ser Ser Lys Ala Asn Gln Gly Arg 50 55 60 Lys Thr Asp Ser Ala Leu Glu Ile Leu Asp Leu Trp Lys Ala Ile Asp 65 70 75 80 Gly Leu Pro Tyr Ser Arg Ser Pro Arg Met Thr Lys Arg Ser Met Gly 85 90 95 Glu Thr Phe Val Lys Pro Arg Thr Gly Asp Leu Arg Ile Val Asp Lys 100 105 110 Asn Val Pro Asp Glu Glu Ala Thr Leu Asn Leu 115 120 <210> 38 <211> 116 <212> PRT <213> Human <400> 38 Met Ala Pro Pro Ser Val Pro Leu Val Leu Leu Leu Val Leu Leu Leu 1 5 10 15 Ser Leu Ala Glu Thr Pro Ala Ser Ala Pro Ala His Arg Gly Arg Gly 20 25 30 Gly Trp Thr Leu Asn Ser Ala Gly Tyr Leu Leu Gly Pro Val Leu His 35 40 45 Leu Pro Gln Met Gly Asp Gln Asp Gly Lys Arg Glu Thr Ala Leu Glu 50 55 60 Ile Leu Asp Leu Trp Lys Ala Ile Asp Gly Leu Pro Tyr Ser His Pro 65 70 75 80 Pro Gln Pro Ser Lys Arg Asn Val Met Glu Thr Phe Ala Lys Pro Glu 85 90 95 Ile Gly Asp Leu Gly Met Leu Ser Met Lys Ile Pro Lys Glu Glu Asp 100 105 110 Val Leu Lys Ser 115 <210> 39 <211> 180 <212 > DNA <213> Rat <400> 39 GCACCTGCTC ACAGGGGACG AGGAGGCTGG ACCCTCAATA GTGCTGGTTA CCTCCTGGGT 60 CCTGTCCTCC ACCTTTCCTC AAAGGCCAAC CAGGGCAGGA AGACAGACTC AGCTCTTGAG 120 ATCCTAGACC TGTGGAAGGC CATAGATGGG CTCCC210 <213> 40 GCACCTGCCC ACCGGGGACG AGGAGGCTGG ACCCTCAATA GTGCTGGCTA CCTTCTGGGT 60 CCCGTCCTCC ACCTTCCCCA AATGGGTGAC CAAGACGGAA AGAGGGAGAC AGCCCTTGAG 120 ATCCTAGACC TGTGGAAGGC CATCGATGGG CTCCCCTACT CCCACCCTCC ACAGCCCTCC 180 <210> 41 <211> 695 <212> DNA <213> Rat <400> 41 AGGACAACTG GGATTACAGA TGTGCATCCC TGCAACCGGC TGCCACACAA GTTCTGGGAT 60 CTGAACTCCT GGCCTCAAAC TTGCCAGCAT TCCTTAGCTG TATGCCGTGC TTTTCCAGTT 120 CCAGGATGGC CTGCTCCAAG CATCTGGTCC TCTTCCTCAC CATCTTGCTA AGCCTCGCAG 180 AAACACCAGA CTCTGCACCT GCTCACAGGG GACGAGGAGG CTGGACCCTC AATAGTGCTG 240 GTTACCTCCT GGGTCCTGTC CTCCACCTTT CCTCAAAGGC CAACCAGGGC AGGAAGACAG 300 ACTCAGCTCT TGAGATCCTA GACCTGTGGA AGGCCATAGA TGGGCTCCCT TATTCCCGCT 360 CTCCAAGGAT GACCAAAAGG TCAATGGGAG AAACGTT TGT CAAGCCGAGG ACTGGAGATC 420 TGCGCATAGT GGACAAGAAT GTTCCGGATG AAGAAGCCAC CCTGAACTTA TAGAGAGTTA 480 GCCCTAGCTC ACTCCTACGT TTCCAGCTCA CCGCCTCTCC CCCCACCCCC GCCCCCAGAA 540 GCGCTCTGAA CACCCTTTCT AAGTCTAAGA CCTTACAACT ATATTCCCTA ATCTCACTAA 600 GACATGTTGT GATATTTAAA GAGTTATTCT GCCCAGCTCC GAAAAAAAAA AAAAAAAAAA 660 AAGAGTTATT CTGACGTAAA AAAAAAAAAA AAAAA 695 <210> 42 <211> 473 <212> DNA < 213> Human <400> 42 GAGGAGCCAG AGAGAGCTGC GGAGAGCTGC CAGCTGCACC GGGCGTGTTC CGCAGCTGTA 60 GGCACCTGTC GTCCTGCCTT CGATGGCTCC TCCCTCCGTC CCCCTGGTCC TCCTCCTCGT 120 CCTCTTGCTG AGCCTGGCAG AGACTCCAGC ATCCGCACCT GCCCACCGGG GACGAGGAGG 180 CTGGACCCTC AATAGTGCTG GCTACCTTCT GGGTCCCGTC CTCCACCTTC CCCAAATGGG 240 TGACCAAGAC GGAAAGAGGG AGACAGCCCT TGAGATCCTA GACCTGTGGA AGGCCATCGA 300 TGGGCTCCCC TACTCCCACC CTCCACAGCC CTCCAAGAGG AATGTGATGG AGACGTTTGC 360 CAAACCAGAG ATTGGAGATC TGGGCATGCT CAGCATGAAA ATTCCCAAGG AGGAAGATGT 420 CCTGAAGTCA TAGATGTCTT CAAATCCCTG TTCCTATCCT TCTTCTCCAG CTC 473 <210> 43 <211> 30 <212> PRT <213> Porcine <40 0> 43 Ala Pro Val His Arg Gly Arg Gly Gly Trp Thr Leu Asn Ser Ala Gly 1 5 10 15 Tyr Leu Leu Gly Pro Val Leu His Pro Pro Ser Arg Ala Glu 20 25 30 <210> 44 <211> 10 <212 > PRT <213> Porcine <400> 44 Cys Ala Pro Ala His Arg Gly Arg Gly Gly 1 5 10 <210> 45 <211> 356 <212> DNA <213> Rat <400> 45 ATGGCTCTGA CTGTCCCTCT GATCGTTCTT GCAGTCCTGC TCAGCCTGAT GGAGTCTCCA 60 GCCTCTGCTC CGGTCCACAG GGGGCGAGGA GGCTGGACCC TCAACAGTGC TGGTTACCTC 120 CTGGGTCCCG TACTCCATCC GCCCTCCAGG GCTGAAGGAG GCGGGAAGGG GAAGACAGCC 180 CTCGGGATCC TGGACCTGTG GAAGGCCATT GATGGGCTCC CCTATCCCCA GTCTCAGTTG 240 GCCTCCAAGA GGAGTCTGGG GGAGACTTTC GCCAAACCAG ACTCTGGAGT AACATTTGTT 300 GGAGTTCCTG ACGTGGTGCC GTGGAAACGA ATCCGACCAG GAACTACGAG GTTTCA 356 <210> 46 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> <400> 46 ATGGCTCTGA CTGTCCCTCT GATCGTTCT 29 <210> 47 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> <400> 47 TGAAACCTCG TAGTTCCTGGTCGGATTCG 29 <210> 48 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> <400> 4 8 AGGCTGGACC CTCAATAGTG CTGGTTAC 28 <210> 49 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> <400> 49 CCATCTATGG CCTTCCACAG GTCTAGGA 28 <210> 50 <211> 126 <212> DNA <213 > Human <400> 50 AGGCTGGACC CTCAATAGTG CTGGTTACCT TCTGGGTCCC GTCCTCCACC TTCCCCAAAT 60 GGGTGACCAA GACGGAAAGA GGGAGACAGC CCTTGAGATC CTAGACCTGT GGAAGGCCAT 120 AGATGG 126 <210> 51 <211> 28 <212> DNA <213> ArtificialTG <220> ACCAAGACGG AAAGAGGG 28 <210> 52 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> <400> 52 GGTCTAGGAT CTCAAGGGCT GTCTCCCT 28 <210> 53 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> <400> 53 GAGGAGCCAG AGAGAGCTGC GGAGAG 26 <210> 54 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> <400> 54 GAGCTGGAGA AGAAGGATAG GAACAGGG 28 <210> 55 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> <400> 55 AGCATATGGC TCCGGTCCAC AGG 23 <210> 56 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> <400> 56 CTGGATCCTC AGGAGGCCAA CTGAGAC 27 <210> 57 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> <400> 57 CAGCCCTCGG CATCCTGGAC C 21 <210> 58 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> <400> 58 GGTCCAGGAT GCCGAGGGCT G 21 <210> 59 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> <400> 59 CTAGACATAT GCCAGCATTG C 21 <210> 60 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> <400> 60 TCGGGCAATG CTGGCATATG T 21 <210> 61 <211> 29 <212> PRT <213> Rat <400> 61 Gly Trp Thr Leu Asn Ser Ala Gly Tyr Leu Leu Gly Pro His Ala Ile 1 5 10 15 Asp Asn His Arg Ser Phe Ser Asp Lys His Gly Leu Thr 20 25 29 <210> 62 <211> 60 <212> PRT <213> Mouse <400> 62 Ala Pro Ala His Arg Gly Arg Gly Gly Trp Thr Leu Asn Ser Ala Gly 1 5 10 15 Tyr Leu Leu Gly Pro Val Leu Pro Val Ser Ser Lys Ala Asp Gln Gly 20 25 30 Arg Lys Arg Asp Ser Ala Leu Glu Ile Leu Asp Leu Trp Lys Ile Ile 35 40 45 Asp Gly Leu Pro Tyr Ser His Ser Pro Arg Met Thr 50 55 60 <210> 63 <211> 117 <212> PRT <213> Mouse <400> 63 Met Ala Cys Ser Val His Leu Val Leu Phe Leu Thr Ile Leu Leu Ser 1 5 10 15 Leu Ala Glu Thr Pro Glu Ser Ala Pro Ala His Arg Gly Arg Gly Gly 20 25 30 Trp Thr Leu Asn Ser Ala Gly Tyr Leu Leu Gly Pro Val Leu Pro Val 35 40 45 Ser Ser Lys Ala Asp Gln Gly Arg Lys Arg Asp Ser Ala Leu Glu Ile 50 55 60 Leu Asp Leu Trp Lys Ile Ile Asp Gly Leu Pro Tyr Ser His Ser Pro 65 70 75 80 Arg Met Thr Lys Arg Thr Met Gly Glu Thr Phe Val lys Ala Asn Thr 85 90 95 Gly Asp Met His Ile Leu Asp Lys Asn Val Pro Lys Glu Glu Ala Thr 100 105 110 Leu Asp Ser Glu Ser 115 <210> 64 <211> 180 <212> DNA <213> Mouse <400> 64 gcacctgctc acaggggacg aggaggctgg accctcaata gtgctggtta cctcctgggt 60 cctgtcctcc ccgtttcctc caaggccgac cagggcagga agagagactc agctcttgag 120 atcctagacc tgtgga <cat> g <attachment> <attachment> GALR2 ligand <400> 65 aggctggacc ctcaatagtg ctggttac 28 <210> 66 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Desig ned oligonucleotide Primer to amplify mouse GALR2 ligand <400> 66 ccatctatgg ccttccacag gtctagga 28 <210> 67 <211> 126 <212> DNA <213> Mouse <400> 67 aggctggacc ctcaatagtg ctggttacct cctgggtcct gtcctccccg tttcctcctagc gagcagcgagc gcgcagc gcgcccagc gagcag ggcag ggcag ggcag agatgg 126 <210> 68 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Designed oligonucleotide Primer for 5 'RACE to amplify mouse GALR2 ligand <400> 68 tccaaggccg accagggcag gaagagag 28 <210> 69 < 211> 28 <212> DNA <213> Artificial Sequence <220> <223> Designed oligonucleotide Primer for 3 'RACE to amplify mouse GALR2 ligand <400> 69 ggtctaggat ctcaagagct gagtctct 28 <210> 70 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Designed oligonucleotide Primer to amplify mouse GALR2 ligand <400> 70 gaaagacagt ctcaaatcaa ggacctgc 28 <210> 71 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Designed oligonucleotide Primer to amplify mouse GALR2 ligand <400> 71 tgagctggaa acgaaggagg agtg aggt 28 <210> 72 <211> 351 <212> DNA <213> Rat <400> 72 atggcctgct ccgtacatct ggtcctcttc ctcaccatct tgctgagcct ggcagaaaca 60 ccggaatctg cacctgctca caggggacga ggaggctgga ccctcaatag tgctggttac 120 ctcctgggtc ctgtcctccc cgtttcctcc aaggccgacc agggcaggaa gagagactca 180 gctcttgaga tcctagacct gtggaagatc attgatggac ttccttattc ccactctcca 240 aggatgacca aaaggacaat gggagaaacg tttgtcaaag cgaatactgg agatatgcac 300 atactggaca agaatgttcc caaggaagaa gccaccctgg actcagagag t 351

[Brief description of the drawings]

Galanin by [Figure 1] ^[35 S] GTPγS binding test,
4 shows the results of detection of the receptor activating action.

FIG. 2 [ ³⁵ S] using a GALR2-expressing cell membrane fraction of the sample fraction obtained in Reference Example 2 (2-3)
The GTPγS binding test results are shown.

FIG. 3 shows the results of analysis of a sample fraction obtained in Reference Example 2 (2-3) using a pig galanin radioimmunoassay kit (Peninsula).

FIG. 4 shows the results of molecular weight analysis of components having a GALR2 activating action ([ ³⁵ S] GTPγS binding promoting activity) obtained in Reference Example 2 (2-4) by gel filtration high performance liquid chromatography.

FIG. 5 shows the results of molecular weight analysis of known peptides by gel filtration high performance liquid chromatography in Reference Example 2 (2-4).

FIG. 6 shows a comparison in amino acid sequence between the peptide of the present invention and a galanin precursor. The amino acid sequence of the swine galanin precursor is shown in the upper part, and the amino acid sequence of the peptide of the present invention is shown in the lower part.

FIG. 7: Ala-Pro-Ala-His-Arg-Gly-Arg-Gly-Gly-Cys
The antibody titer of the mouse immunized with the (-NH ₂ ) -KLH complex was determined by HR
P-labeled Ala-Pro-Ala-His-Arg-Gly-Arg-Gly-Gly-Cys-NH
₂ shows the results of an examination using the method.

FIG. 8: Ala-Pro-Ala-His-Arg-Gly-Arg-Gly-Gly-Cys
1 shows a typical example of screening for a hybridoma after cell fusion using a mouse immunized with a (-NH ₂ ) -KLH complex.

FIG. 9: Ala-Pro-Ala-His-Arg-Gly-Arg-Gly-Gly-Cys
Ala-Pro-Ala-His-Arg-Gly-Ar of monoclonal antibody GR2-1N prepared using (-NH ₂ ) -KLH complex as an immunogen
The reactivity to g-Gly-Gly-Cys-NH ₂ (amide of SEQ ID NO: 44), porcine ligand peptide (1-60) (SEQ ID NO: 31) and rat galanin (SEQ ID NO: 61) was HRP-labeled. Ala-Pro-Ala-His-Arg-Gly-Arg-Gly-Gly-
The results obtained by a competition method using Cys-NH ₂ -EIA are shown.

FIG. 10 shows a diagram relating to a method for preparing a structural gene of a porcine ligand peptide described in Reference Example 20.

FIG. 11 shows a construction diagram of a fusion protein expression vector described in Reference Example 21.

FIG. 12 shows a construction diagram of a pig-type ligand peptide expression strain described in Reference Example 21.

FIG. 13 shows an analysis result (electropherogram) using SDS-polyacrylamide gel electrophoresis described in Reference Example 23.

14 shows a diagram illustrating the results of a feeding test performed in Reference Example 24. FIG.

FIG. 15 shows blood LH (a), FSH (b), and GH 10 minutes after administration of rat GALP into rat third ventricle.
(C), TSH (d), ACTH (e) and Prolacti
The n (f) concentration fluctuation is shown. Each concentration was shown as a measured value ± standard error. ** indicates that there is a significant difference at the significance level of 1% (n = 6-7).

FIG. 16 shows a change in blood LH concentration when rat GALP and galanin were administered into a rat ventricle. In the figure,-△-is control,-○-is rat GALP.
1 nmol administration,-●-indicates rat GALP 5 nmol
Administration,-*-shows the results of administration of 5 nmol galanin. The concentration of LH was shown as a measured value ± standard error. Also,*
* Indicates that there is a significant difference at the significance level of 1% (n = 5).

[FIG. 17] Blood L when rat GALP was intracerebroventricularly administered to Zucker fatty rats and Zucker lean rats.
The fluctuation of H concentration is shown. In the figure,-△-indicates control when administered intracerebroventricularly to Zucker lean rats, and-○-indicates Zu.
Control when administered intracerebroventricularly to cker fatty rats,-▲-indicates rat GALP (1 nmol) when intravenously administered to Zucker lean rats,-●-indicates Zucker f
Rat GALP when administered intraventricularly to atty rats
(1 nmol) shows the results. The concentration of LH was shown as a measured value ± standard error. ** indicates that there is a significant difference at the significance level of 1% (n = 5). * Indicates that there is a significant difference at the significance level of 5% (n = 5).

FIG. 18: Pig small intestinal galanin (galanin), pig type G
alR2 ligand (P-GALR2L), rat type Ga
1R2 ligand (R-GALR2L), human GalR
FIG. 2 is a diagram comparing the amino acid sequences of the two ligands (H-GALR2L) and the mouse GalR2 ligand (M-GALR2L) of the present invention.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI theme coat ゛ (Reference) A61P 5/08 A61P 13/08 13/08 15/08 15/08 15/12 15/12 35/00 35 / 00 C07K 7/06 C12N 15/09 ZNA 14/00 // C07K 7/06 A61K 37/43 14/00 C12N 15/00 ZNAA (72) Inventor Tetsuya Otaki 1-7-9 Kasuga 9, Tsukuba, Ibaraki, Japan Takeda Kasuga Heights 802 F-term (reference) 4B024 AA01 BA80 CA04 DA06 EA04 GA11 4C084 AA02 AA13 BA01 BA08 BA17 BA20 BA24 CA18 CA28 DB09 NA14 ZA701 ZA811 ZB261 ZC011 ZC041 ZC042 ZC111 ZC521 4C087 BC83 ZA12 Z14A14 BA15 BA20 CA45 EA20 FA33 FA73 FA74

Claims (22)

[Claims] Claims: 1. A galanin-like peptide (Galanin-like pep)
tide (GALP)) or a salt thereof, or a luteinizing hormone (LH) secretion regulator containing DNA encoding a galanin-like peptide. 2. The LH secretion regulator according to claim 1, wherein the galanin-like peptide is a peptide having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 35. 3. The galanin-like peptide is SEQ ID NO: 31,
The LH secretion regulator 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 33, 34 or 62. 4. A DNA encoding a galanin-like peptide
Is a DNA containing a nucleotide sequence that hybridizes under high stringent conditions to the nucleotide sequence represented by SEQ ID NO: 32, 39, 40, or 64. 5. The L according to claim 1, which is an LH secretagogue.
H secretion regulator. 6. The L according to claim 1, which is an LH secretion inhibitor.
H secretion regulator. 7. The LH according to claim 5, which is a preventive or therapeutic agent for infertility, irregular menstruation, dysmenorrhea, amenorrhea, rare menstruation, premenstrual syndrome, menopause, pituitary dysfunction or obesity. Secretagogue. 8. The LH secretion inhibitor according to claim 6, which is a preventive / therapeutic agent for prostate cancer, benign prostatic hyperplasia, precocious puberty or LH-producing pituitary tumor. 9. An LH secretion regulator containing a galanin-like peptide agonist. 10. An LH secretion regulator comprising a galanin-like peptide inhibitor. 11. A galanin-like peptide (Galanin-like p
eptide (GALP)) or a salt thereof, or a luteinizing hormone-releasing hormone (LHRH) secretion regulator containing DNA encoding a galanin-like peptide. 12. The galanin-like peptide is SEQ ID NO: 35
12. The LHRH secretion regulator according to claim 11, which is a peptide having the same or substantially the same amino acid sequence as the amino acid sequence represented by: 13. The galanin-like peptide is SEQ ID NO: 3.
The LHRH secretion regulator according to claim 11, which is a peptide containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by 1, 33, 34 or 62. 14. DN encoding a galanin-like peptide
The LHRH secretion regulator according to claim 11, wherein A is a DNA containing a nucleotide sequence that hybridizes under high stringent conditions to the nucleotide sequence represented by SEQ ID NO: 32, 39, 40 or 64. 15. An LHRH secretion promoter.
The LHRH secretion regulator according to the above. 16. The method according to claim 11, which is an LHRH secretion inhibitor.
The LHRH secretion regulator according to the above. 17. The LHRH according to claim 15, which is an agent for preventing or treating infertility, irregular menstruation, dysmenorrhea, amenorrhea, rare menstruation, premenstrual syndrome, menopause, pituitary dysfunction or obesity. Secretagogue. 18. The LHRH secretion inhibitor according to claim 16, which is a prophylactic / therapeutic agent for prostate cancer, benign prostatic hyperplasia, precocious puberty or LHRH-producing pituitary tumor. 19. An LHRH secretion regulator containing a galanin-like peptide agonist. 20. An LHRH secretion regulator containing a galanin-like peptide inhibitor. 21. A galanin-like peptide (Galanin-like peptide) for producing an LH secretion regulator or an LHRH secretion regulator.
peptide (GALP)) or a salt thereof, or a DNA encoding a galanin-like peptide. 22. A pharmacologically effective amount of a galanin-like peptide (GA) for a mammal.
LP)) or a salt thereof, or a method for preventing or treating LH secretion dysregulation or LHRH secretion dysregulation, which comprises administering DNA encoding a galanin-like peptide.