WO2003002603A1

WO2003002603A1 - Novel neuropeptide y-like peptide

Info

Publication number: WO2003002603A1
Application number: PCT/JP2002/006397
Authority: WO
Inventors: Shunichiro Matsumoto; Jun Takasaki; Tetsu Saito; Masazumi Kamohara; Takatoshi Soga; Masashi Ukai
Original assignee: Yamanouchi Pharmaceutical Co., Ltd.
Priority date: 2001-06-27
Filing date: 2002-06-26
Publication date: 2003-01-09
Also published as: JPWO2003002603A1

Abstract

A novel NPY-like peptide; a polynucleotide encoding it; and a process for producing the same.

Description

Yoshiaki Meshiro New neuropeptide Y-like peptide Technical field

The present invention relates to a novel neuropeptide Y-like peptide. Background art

Neuropeptide Y (hereinafter abbreviated as NPY) Family 1 is a bioactive peptide composed of three types: NPY, peptide YY (PYY), and knee peptide (pancreaticpolypeptide; PP). It is a group (Pgu pt., 62, 1—11, 1996 in Regu). All peptides are 36 amino acids and are C-terminally amidated linear peptides.

NPY was isolated and purified from pig brain in 19982 and subsequent studies have shown that it is the most abundant neuropeptide in the mammalian central and peripheral nervous system (Nature, 296). , 659-60, 1 982). NPY expression is found in the central nervous system (especially the hypothalamus, hippocampus, cerebral cortex, and brainstem), adrenal medulla, and parasympathetic nerve.

PYY is also a bioactive peptide isolated and purified from pig small intestine in 1982, the same as NPY (Proc. Natl. Ac ad. Sc, USA, 79, 25 14-8, 1 982). PYY has 70% homology to NP Y in the primary amino acid sequence, and its expression has been confirmed in the lower small intestine, large intestine, and knee.

ΡΡ was identified in 1975 as the first molecule in the インスリン family as a byproduct of the chicken insulin purification process (jBiol. Chem., 25

0, 9369-76, 1 975). Although 存在 Ρ Υ and Ρ Υ Υ have been identified in all vertebrates, Ρ Ρ is thought to have occurred early in the evolution to tetrapods. It has amino acid sequence homology, and its expression is mainly observed in mucus.

ΝΡΥThe gene is present on human chromosome 7 ρ15.1. ΡΥΥ gene And the PP gene are all linked to chromosome 17q21.1 in human at a distance of only 10 kb. From molecular evolutionary analysis, the PP gene is replicated as a copy of the PYY gene. geneduplication). NPY is the most conserved molecule in molecular evolution among known neuropeptides. In the known ortholog, 22 out of 36 amino acids are identical. On the other hand, PYY has less than 15 amino acids conserved across species, whereas PP has only 7 identical amino acids. Based on these facts, PYY is considered to be a molecule that evolves faster than NPY, and it has been suggested that the replication of the PP gene than the PYY gene was one of the factors that accelerated the evolution rate of PYY. .

In the N PY family, three proline residues and two tyrosine residues, which are commonly held in the amino acid primary sequence, are (polyproline helix) 1 (turn) 1

(Helix) and a highly flexible C-terminal 4 amino acid tertiary structure are formed. This structure is called ΓΡP-foId ”and is thought to play an important role in expressing the activity of the NPY family (丄 Biol.Chem., 265, 11706-1). 2, 1 990).

Functional expression of the NPY family occurs through binding to Ν Ν -specific receptors (Trends Neurosc, 20, 294—8, 1997). It is known that there are five different subtypes of the NPY family so far, and G protein-coupled receptor (GPCR) Y1, Υ2, Υ4, Υ5, and Υ6 are at the gene level. Has been identified. The Υ1 receptor has the highest homology, at 42% (in terms of amino acid sequence), to the Υ4 receptor, and 51% homology to the 遺伝子 6 receptor, a pseudogene that has no function in humans. Interestingly, the Υ1 receptor has only 35% and 31% homology to the Υ5 and Υ2 receptors, respectively, in amino acid sequence. These facts indicate that ΝΡΥ receptors are the least intersubtype homologous group in the GPCR superfamily. All five ΝΡΥ receptors activate the intracellular signaling system through the activation of the pertussis toxin-sensitive G protein, and suppress the accumulation of intracellular cAMP. In addition, Y1 and Y2 receptors cause an increase in intracellular Ca concentration upon activation, and Y2 receptors further cause K channel activation It has also been reported.

Physiological activities of NPY have been suggested to be involved in eating, sexual behavior, peripheral movement, and blood pressure. Of these physiological functions, the best studied is related to feeding during central administration, and a series of studies have reported that NPY is the most effective food-stimulating substance (B ioch em. Ce I to B iol., 78, 371 -92, 2000). When NPY is administered intravenously into rats, an increase in food intake is observed, which eventually leads to weight gain and obesity. Before daily eating, NPY expression increases in the paraventricular nucleus of the hypothalamus, which is the center of satiety (Am. J. Physiol., 270 (4 Pt1), E589—95, 1996). ). In starvation, NPY expression in the hypothalamus is increased, but its expression is attenuated by eating

(Am. J. Physiol., 266 (5 Pt2), R1 687-91, 1994). Interestingly, however, パターンノ knockout mice (Κ Ο mice) did not change their feeding patterns (Natur β, 381, 41 5-21, 1 996), indicating that ο bob mice and Ν Ρ Υ When crossed with mice, strong weight loss is observed, accompanied by reduced food intake and increased energy metabolism (Science, 274, 1704-7, 1996).

PYY and PP are also known to cause hyperphagia. It has been reported that PYY significantly increases the amount of food consumed by intraventricular administration to rats, and that the effect is more remarkable than that of NPY [Am. J. Physio, 259 (2 Pt 2), R 31 7-23, 1 990, Rain Re s., 341, 200-3, 1985, Rain Re s., 805, 20-8, 1 998, and Pysio to Beha v., 58 , 731—5, 1 995]. PP is known to stimulate feeding by intraventricular administration [Am. J. Physiol., 269

(5 Pt2), R983-7, 1995]. PYY and PP are also thought to regulate gastrointestinal tract functions and peripheral physiological functions such as excretion of the knee. PYY is known to suppress gastric acid secretion in many animal species, and has antisecretory activity in rat small intestine. Conversely, stomach acid increases PYY mRNA transcription. In the knee, PYY is stored together with glucagon in secretory granules of cells, and PYY has a glucose-stimulated inhibitory activity on insulin secretion (Acta Pysiol. Scand., 157, 305-6, 1996). ). PP, gastric acid secretion J. Physiol., 269 (5Ρ2), R983-7, 1995].

Furthermore, anti-obesity drugs are being studied for Ν Ρ Υ family inhibitors such as 1229 U91, BMS-192558, J-104870, and BIBP 3226 (J. Pharma co I. Exp. Ther., 275, 1 26 1—6, 1 995, J. An tibiot. (Tokyo), 48, 1 055— 9, 1 995, Biooch em. Biophy s. Res. Commun., 266, 88-91, 1999, and J. Pharma coI. Exp. Ther., 275, 136-42, 1995).

As mentioned above, the NPY family is known to be useful as a new drug target. Disclosure of the invention

An object of the present invention is to provide a novel NPY-like peptide, a polynucleotide encoding the same, and a method for producing them.

As a result of intensive studies, the present inventors have obtained a polynucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 1, and the peptide encoded by the polynucleotide has the highest homology to PYY belonging to the NPY family. Was found to have. And, like PYY, this peptide was expressed in the hypothalamus, which controls the feeding function, the small intestine, which controls gastric acid secretion, and the pituitary gland, which controls the insulin secretion function. The present invention has been clarified to be a novel NPY-like peptide having a function, and the present invention has been completed.

That is, the present invention

[1] a peptide consisting of the amino acid sequence represented by SEQ ID NO: 3;

[2] a peptide consisting of the amino acid sequence represented by SEQ ID NO: 2;

[3] a polynucleotide encoding the peptide of [1] or [2];

[4] An expression vector containing the polynucleotide according to [3];

[5] a transformant comprising the polynucleotide of [3];

[6] The method for producing the peptide according to [1] or [2], comprising a step of culturing the transformant according to [5], and a step of collecting the peptide; [7] It can be produced by a method comprising a step of culturing a transformed eukaryotic cell containing a polynucleotide encoding a peptide consisting of the amino acid sequence represented by SEQ ID NO: 2, and a step of recovering the peptide. Petit

About.

In addition, WO 0168050 published after the priority date of the present application merely describes the amino acid sequence represented by SEQ ID NO: 2 and the base sequence encoding the same. There is no specific method for obtaining a peptide consisting of the amino acid sequence represented by SEQ ID NO: 2 or a polynucleotide encoding the same, and there is no example in which the above-mentioned peptide or polynucleotide was obtained. There is no statement to support the acquisition of the peptide, etc. Further, the amino acid sequence represented by SEQ ID NO: 3 has not been disclosed or suggested. That is, the peptide consisting of the amino acid sequence represented by SEQ ID NO: 3 or SEQ ID NO: 2, and the polynucleotide encoding these were obtained by the present inventors for the first time. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is an explanatory diagram showing the results of an alignment analysis between the amino acid sequence of the peptide of the present invention and the amino acid sequences of three types of known peptides belonging to the NPY family. BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be described in detail.

[11 Polypeptide of the Present Invention

The peptides of the present invention include:

(1) a peptide consisting of the amino acid sequence represented by SEQ ID NO: 3; and

(2) a peptide consisting of the amino acid sequence represented by SEQ ID NO: 2

Is included.

“Peptide consisting of the amino acid sequence represented by SEQ ID NO: 3”, which is one of the peptides of the present invention, is a human-derived peptide consisting of 42 amino acid residues. The above-mentioned “peptide consisting of the amino acid sequence represented by SEQ ID NO: 3” is referred to as “peptide consisting of the amino acid sequence represented by SEQ ID NO: 2” as shown in Example 1 below. The translated precursor is considered to be a mature form in which the N-terminal signal peptide (that is, the peptide consisting of amino acids 1 to 28 in the amino acid sequence represented by SEQ ID NO: 2) has been cleaved off. .

The peptide of the present invention has a function similar to that of PYY, specifically, a feeding regulation function (preferably, a feeding enhancement function), a gastric acid secretion suppressing function, or an insulin secretion suppressing function.

The method for confirming whether or not a certain peptide exhibits the “feeding regulation function” is not particularly limited, but for example, it can be confirmed by the following method. That is, the test peptide is administered in an appropriate amount (for example, 1 mg, 2 mg, 5 mg, and 10 mg each) into the rat ventricle, and after a predetermined time has passed since the administration (for example, 30 minutes, 60 minutes) After 90 minutes, after 120 minutes, and after 240 minutes), the amount of food consumed is calculated from the decrease in total food consumption. As a result, if a change in the rat intake in a test peptide dose-dependent manner is observed, it can be determined that the test peptide has a food intake regulating function. For example, if an increase in rat food intake is observed in a test peptide dose-dependent manner, it can be determined that the test peptide has a food intake enhancing function.

(Brain Res., 341, 200-3, 1985, and Am. J. PysioI., 259, R317-23, 1990).

C21 polynucleotide of the present invention

The polynucleotide of the present invention is not particularly limited as long as it is a polynucleotide encoding the peptide of the present invention.

The polynucleotide of the present invention comprises a sequence consisting of nucleotides 117 to 248 in the nucleotide sequence represented by SEQ ID NO: 1. In the polynucleotide or the nucleotide sequence represented by SEQ ID NO: 1, A polynucleotide consisting of a sequence consisting of the 36th to 248th bases is preferred. The former polynucleotide encodes a peptide consisting of the amino acid sequence represented by SEQ ID NO: 3, and the latter polynucleotide encodes a peptide consisting of the amino acid sequence represented by SEQ ID NO: 2. As used herein, the term “polynucleotide” includes both DNA and RNA.

The method for producing the polynucleotide of the present invention is not particularly limited. Examples of the method include (a) a method using the polymerase chain reaction (PCR), and (b) a conventional method of inheritance. A method using a child engineering technique (ie, a method of selecting a transformant containing the desired cDNA from a transformant transformed with the cDNA library), or (c) a chemical synthesis method. Can be mentioned. Hereinafter, each manufacturing method will be sequentially described.

In the method using PCR [the above-mentioned production method (a)], the polynucleotide of the present invention can be produced, for example, by the following procedure.

That is, mRNA is extracted from human cells or tissues capable of producing the peptide of the present invention. Next, based on the nucleotide sequence of the polynucleotide encoding the peptide of the present invention, a pair of two primer sets that can sandwich the entire length of mRNA corresponding to the peptide of the present invention, or a part of the mRNA Create a pair of primer sets that can sandwich the A region. The reverse transcriptase-polymerase chain reaction (RT-PCR) is performed by appropriately adjusting the reaction conditions (eg, denaturation temperature or denaturant addition conditions) to obtain the full-length cDN encoding the peptide of the present invention. A or a part of it can be obtained.

Further, from mRNA prepared from human cells or tissues having the ability to produce the peptide of the present invention, cDNA prepared using reverse transcriptase or commercially available human cells or tissue-derived cDNA can be used. By performing PCR as a type III, full-length cDNA or a part thereof encoding the peptide of the present invention can also be obtained. More specifically, first, total RNA including mRNA encoding the peptide of the present invention is extracted from a cell or tissue capable of producing the peptide of the present invention by a known method. Examples of the extraction method include a guanidine thiocyanate hot phenol method, a guanidine thiocyanate-guanidine hydrochloride method, and a guanidine thiocyanate cesium chloride method. It is preferable to use the cesium method. Cells or tissues having the ability to produce the peptide of the present invention include, for example, a Northern blotting method using a polynucleotide encoding the peptide of the present invention or a part thereof, or a peptide specific to the peptide of the present invention. It can be identified by Western blotting using an antibody.

Subsequently, the extracted mRNA is purified. Purification of mRNA can be performed according to a conventional method.For example, mRNA is adsorbed onto an oligo (dT) cellulose column and then eluted. Can be purified. If desired, mRNA can be further fractionated by sucrose density gradient centrifugation or the like. Also, without extracting mRNA, commercially available extracted and purified mRNA can also be used.

Next, the purified mRNA is subjected to a reverse transcriptase reaction in the presence of, for example, a random primer, an oligo dT primer, and / or a custom-synthesized primer to synthesize first-strand cDNA. This synthesis can be performed by a conventional method. Using the obtained first-strand cDNA, PCR can be performed using two types of primers sandwiching the full length or a partial region of the target polynucleotide to amplify the target cDNA. The obtained DNA is fractionated by agarose gel electrophoresis or the like. If desired, the DNA fragment can be obtained by cleaving the DNA with a restriction enzyme or the like and connecting it. Also, a desired DNA fragment can be obtained from genomic DNA.

In the method using the conventional genetic engineering technique [the above-mentioned production method (b)], the polynucleotide of the present invention can be produced, for example, by the following procedure.

First, using the mRNA prepared by the above-described PCR as a type I, a single-stranded cDNA was synthesized using a reverse transcriptase, and then a double-stranded cDNA was synthesized from the single-stranded cDNA. Combine A. Examples of the method include the S1 nuclease method (E fstratiadis, A. et al., Cell, 7, 279-288, 1976), and the Land method (L and, H. et al., Nucleic Acid Res Res. , 9, 2251-1 2266, 1 981), 0. Joon Yoo method (Yoo, OJ et al., Proc. Natl. Ac a d. Sc, USA, 79, 1049-1053, 1 9 83) or the Okay ama-Berg method (Okayama, H. and Berg, P., Mo for Cell to Biol., 2, 161-170, 1982), etc. Can be.

Next, after preparing a recombinant plasmid containing the double-stranded cDNA, the recombinant plasmid is introduced into Escherichia coli (for example, DH5a strain, HB101 strain, or JM109 strain) for transformation, for example, tetracycline, ampicillin Recombinants are selected based on drug resistance to, or kanamycin. For example, when the host cell is Escherichia coli, the transformation of the host cell is carried out by the method of Hanahan (Hanahan, DJ, Mo to Bio, 166, 557-580, 1983), ie, C a CI ₂ , M The method can be carried out by adding the recombinant DNA to the above-described recombinant cells prepared in the presence of gC I ₂ or Rb CI. Also, commercially available competent cells can be used. As a vector, a phage vector such as a lambda-based one can be used other than the plasmid.

Methods for selecting a transformant having the desired cDNA from the thus obtained transformants include, for example, the following (1) screening method using a synthetic oligonucleotide probe, or (2) A method using a screening method using a probe prepared by PCR can be adopted.

Screening method using synthetic oligonucleotide probe [Selection method described above]

In (1)], for example, a transformant having the desired cDNA can be selected by the following procedure.

That is, an oligonucleotide corresponding to all or a part of the peptide of the present invention was synthesized, and this was used as a probe (labeled with ³² P or ³³ P). After hybridization, search for the obtained positive strain and select it. When synthesizing an oligonucleotide for a probe, a nucleotide sequence derived using codon usage can be used, or a plurality of nucleotide sequences obtained by combining possible nucleotide sequences can be used. it can. In the latter case, the type can be reduced by including inosine.

Screening method using a probe prepared by PCR [Selection method described above]

In (2)], for example, a transformant having the desired cDNA can be selected by the following procedure.

That is, oligonucleotides of sense primers and antisense primers corresponding to a part of the peptide of the present invention are synthesized, PCR is performed by combining these, and a DNA fragment encoding all or a part of the target peptide To amplify. As the type I DNA used here, cDNA or genomic DNA synthesized by reverse transcription reaction from mRNA of the cell producing the peptide of the present invention can be used. The DNA fragment thus prepared is labeled with, for example, ³² P or ³³ P, and used as a probe for colony hybridization or plaque hybridization to obtain the desired DNA fragment. Select a transformant with cDNA. A method for collecting the polynucleotide of the present invention from the obtained desired transformant can be obtained by a known method (for example, Sambrook, J. et al., "Molecular CI on ng—AL aboratory Manual, Cold). For example, a fraction corresponding to the plasmid DNA is separated from the cells, and the cDNA region is separated from the obtained plasmid DNA. It can be done by cutting out.

In the method using the chemical synthesis method [the above-mentioned production method (c)], for example, the polynucleotide of the present invention can be produced by binding the DNA fragment produced by the chemical synthesis method. Each DNA should be synthesized using a DNA synthesizer [for example, Oligo 1 000M DNA Synthesizer (manufactured by Beckman), or 394 DNA RNA Synthesizer (manufactured by App Ied Biosystems)] Can be.

In addition, the polynucleotide of the present invention can be prepared based on the information of the peptide of the present invention, for example, by the phosphite triester method (Hunkapi IIer, M. et al., Nature, 10: 105-111, 1984), etc., and can be produced by chemical synthesis of nucleic acids. The codon for the desired amino acid is known per se and may be arbitrarily selected. For example, it can be determined according to a conventional method in consideration of the codon usage of the host to be used (Crantham, R., et al. Nucleic A cids Res., 9, r 43—r 74, 198 1). Further, partial modification of the codons of these nucleotide sequences can be performed by a site-directed mutagenesis method using a primer comprising a synthetic oligonucleotide encoding the desired modification (Mark, D. r. Et al., Proc. Natl. Acad. Sc, USA, 81, 5662-5666, 1984).

The sequencing of DNA obtained by the various methods described so far can be carried out, for example, by the chemical modification method of Maxam-Gilbert (Maxam, AM and Gibert, W., "Methodsin Enzymo Iogy"). , 65, 499—55 9, 1980) Didoxynucleotide chain termination method (Messing, J. and Vieira, J., Gene, 19, 269-276, 1982), etc. Can be.

Γ31 Expression vector, transformant, and peptide production method of the present invention

Host cells (including eukaryotic and prokaryotic host cells) can be transformed by reintegrating the isolated polynucleotide of the present invention into an appropriate vector DNA. By introducing an appropriate promoter and a sequence related to expression into these vectors, the polynucleotide can be expressed in each host cell.

For example, eukaryotic host cells include cells such as vertebrates, insects, and yeast. Examples of vertebrate cells include monkey COS cells (GIuzman, Y., Ce). ll, 23, 175-182, 1981), a dihydrofolate reductase-deficient strain of Chinese hamster ovary cells (CHO) (UrI aub, G. and Cansin, LA, Proc. Natl. Sc, USA, 77, 421 6-4220, 1980), human embryonic kidney-derived HEK 293 cells, and 293-EBNA in which the EBN A-1 gene of Epstein-Barr virus was introduced into the HEK 293 cells. Cells (Invitrogen) and the like.

As a vertebrate cell expression vector, a vector having a promoter, an RNA splice site, a polyadenylation site, a transcription termination sequence, and the like located upstream of a polynucleotide to be normally expressed can be used. Furthermore, if necessary, it may have a replication origin. Examples of the expression vector include, for example, pSV2 dhfr having an early promoter of SV40 (Subbramani, S. et al., Mo: C CI: Biol., 1, 854-864, 19981); PEF-BOS (Mizushi ma, S. and Nagata, S., Nucleic Acids Res., 18, 53, 22, 1990) having a human elongation factor promoter and a cytomegalovirus promoter p CEP 4 (Invitrog _θn company) and the like.

When COS cells are used as host cells, they have an SV40 origin of replication as an expression vector, are capable of autonomous growth in COS cells, and have a transcription promoter, a transcription termination signal, and an RNA splice site. For example, pME18S (Maruyama, K. and Takebe, Y., Med.Immuno I., 20, 27-32, 1990), pEFBOS (Mizushima, S. and Nagata, S., Nucleic A cids Res,, 18). 5322, 1990), or pCDM8 (Seed, B., Nature, 329, 840-842, 1987).

The expression vector is, for example, a DEAE-dextran method (Lutman, H. and Magnusson, G., Nucleic Acids Res., 11, 11, 295- 1308, 1983), calcium phosphate-D. Ν coprecipitation method (Grah am, FL.L. and vander Ed, AJ, Viro Iogy, 52, 456-457, 1973), commercially available transfection reagents (for example, FuGENE ^TM 6T ransfection reagent (Roc β Diagnostics), or electric pulse perforation (Neumann, E. et al., EMBO J. 1, 841-845, 1982). It can be taken up by cells.

When a CHO cell is used as a host cell, a vector capable of expressing a neo gene functioning as a G418 resistance marker together with an expression vector containing a polynucleotide encoding the peptide of the present invention, for example, p RSVn eo (S amb roo K, J. b, Mo lecular Cloning— A

La boratory Manual, Cold Spring Harbor, NY, 1989) or p SV 2—n β o (So uth rnrn, P.J. and Berg, P., J. Mo. , 1, 327-341, 1982), and by selecting a G418-resistant colony, transformed cells capable of stably producing the peptide of the present invention can be obtained. Obtainable.

When 293-EBNA cells are used as host cells, PCEP4 (Invitrogen, Inc.), which has an Epstein-Barr virus replication origin as an expression vector and is capable of self-replication in 293- £ 8 cells ) Can be used.

The transformant of the present invention can be cultured according to a conventional method, and the culture produces the peptide of the present invention extracellularly. A medium that can be used for the culture Alternatively, various commonly used media can be appropriately selected depending on the host cell employed. For example, in the case of COS cells, a medium such as RPMI-1640 medium or Dalbecco's Modified Eagle's Minimum Essential Medium (DMEM) may be added to a medium such as fetal bovine serum (FBS) if necessary. A medium to which the components have been added can be used. In the case of 293-EBN A cells, use a medium such as Dulbecco's Modified Eagle's Minimum Essential Medium (DMEM) supplemented with serum components such as fetal calf serum (FBS) plus G418. it can.

By culturing the transformant of the present invention, the peptide of the present invention produced outside the cells of the transformant can be produced by various known methods utilizing the physical properties, biochemical properties, and the like of the peptide. Separation and purification can be performed by this separation operation method. Specifically, the culture solution containing the peptide of the present invention is treated with, for example, a usual protein precipitant, ultrafiltration, various liquid chromatography [eg, molecular sieve chromatography (gel filtration), adsorption chromatography, ion The peptide of the present invention can be purified by exchanger chromatography, affinity chromatography, high performance liquid chromatography (HP LC) or the like, or dialysis, or a combination thereof.

The expression and fusion of the peptide of the present invention can be facilitated by expressing the peptide of the present invention by fusing it in-frame with a marker sequence. Examples of the marker sequence include FLAG epitope, hexahistidine tag, hemagglutinin tag, and my cepitope. In addition, by inserting a specific amino acid sequence recognized by a protease (eg, enterokinase, factor Xa, or thrombin) between the marker sequence and the peptide of the present invention, the marker sequence portion can be inserted. These proteases can be cleaved and removed.

The peptide of the present invention can be produced by, for example, a chemical synthesis method using an automatic peptide synthesizer in addition to the production method using the transformant of the present invention as described above. Automatic peptide synthesizers include, for example, Applied Biosystems 43OA, Millipore 9050, and Shimadzu PSSM-8, etc. Solid phase synthesis for binding can be performed. In this method, the amino group The Boc method, which uses cleavage of the protecting group (Bo. Group) with trifluoroacetic acid, and the Fmoc method, which removes the Fmoc group at the N-position with piperidine, are both known. After removal, it can be purified by high-performance liquid chromatography, etc. [Omi, Tsujimura, Inagaki, Ed., Cell Engineering Separate Volume, Anti-Peptide Antibody Experimental Protocol J p 25-46 (1 994) Shujunsha] .

An antibody that reacts with the peptide of the present invention (for example, a polyclonal antibody or a monoclonal antibody) can be obtained, for example, by directly administering the peptide of the present invention or a fragment thereof to various animals. Using a plasmid into which a polynucleotide encoding the peptide of the present invention has been introduced, a DNA vaccine method (Raz, E. et al., Proc. Natl. Acad. Sc, USA, 91, 951 9 -9523, 1994; or Donnel Iy, J. J. et al., J. Infect. Diss., 173, 314-320, 1996).

The polyclonal antibody is sensitized, for example, by immunizing an emulsion obtained by emulsifying the peptide of the present invention or a fragment thereof in a suitable adjuvant (for example, Freund's complete adjuvant) into the peritoneal cavity, subcutaneous region, or vein. It can be produced from the serum or eggs of a purified animal (eg, egret, rat, goat, or chick). From the thus-produced serum or egg, a lipo-specific antibody can be separated and purified by a conventional protein isolation and purification method. Examples of such separation and purification methods include centrifugation, dialysis, salting out with ammonium sulfate, and chromatography using DEAE-cellulose, hydroxyapatite, protein A agarose, or the like.

Monoclonal antibodies can be easily prepared by those skilled in the art, for example, by the cell fusion method of Koehler and Milstein (Koh IΘr, G. and M I Istein, C., Nature, 256, 495-497, 1975). It is possible to manufacture.

That is, an emulsion obtained by emulsifying the peptide of the present invention or a fragment thereof in an appropriate adjuvant (for example, complete Freund's adjuvant) is repeatedly inoculated into the peritoneal cavity, subcutaneous, or vein of a mouse several times every several weeks. Immune by. After the final immunization, spleen cells are removed and fused with myeloma cells to produce hybridomas. Myeoma cells for obtaining hybridomas include, for example, hypoxanthine-guanine-phosphoribosyltransferase deficiency or thymidine kinase deficiency. Myeloma cells having a marker such as loss (eg, mouse myeloma cell line P 3 X63Ag 8.U 1) can be used. Further, as the fusion agent, for example, polyethylene glycol can be used. Further, as a medium for producing hybridomas, for example, a commonly used medium such as Eagle's minimum essential medium, Dulbecco's modified minimum essential medium, or RPMI-1640 is used, for example, 10 to 300/300. 0 fetal fetal serum can be added and used as appropriate. Fusion strains can be selected by the HAT selection method. The screening of hybridomas is performed by using well-known methods such as ELISA or immunohistochemical staining using the culture supernatant, and the clones of hybridomas secreting the desired antibody can be selected. In addition, by repeating subcloning by the limiting dilution method, the monoclonality of the hybridoma can be guaranteed. The high-purity doma obtained in this way can be purified in a medium that can be purified for 2 to 4 days or in the abdominal cavity of a BALBBZc mouse pretreated with pristane for 10 to 20 days. Antibodies can be produced.

The monoclonal antibody thus produced can be separated and purified from the culture supernatant or ascites by a conventional protein isolation and purification method. Examples of such separation and purification methods include, for example, centrifugation, dialysis, salting out with ammonium sulfate, or chromatography using DEA-cellulose, hydroxyapatite, or protein A agarose.

In addition, an antibody fragment containing a monoclonal antibody or a part thereof may be obtained by incorporating all or a part of the polynucleotide encoding the monoclonal antibody into an expression vector, and preparing an appropriate host cell (for example, Escherichia coli, yeast, or animal cell). ) Can be introduced for production.

Antibodies (including polyclonal antibodies and monoclonal antibodies) separated and purified as described above are digested with a peptidase (for example, pepsin or papain) by a conventional method, and then a conventional protein isolation is performed. An antibody fragment containing a part of the active antibody, for example, F (ab ') ₂ , Fab, Fab', or Fv can be obtained by separation and purification by a purification method.

Furthermore, an antibody that reacts with the peptide of the present invention can be obtained by the method of Cracson et al. Or the method of Zebede et al. (CI ackson, T. et al., Nature, 352, 624-628, 1 991; or Zebedee, S. et al., In Proc. N at Ac a d. Sc in USA, 89. 31 75-31 79, 1992), a single strand (sίng I echain) It can also be obtained as Fv or Fab. Human antibodies can also be obtained by immunizing transgenic mice (Lonberg, N. et al., Nature, 368, 856-859, 1994), in which the mouse antibody gene is replaced with a human antibody gene. It is possible.

By using the peptide of the present invention, it is possible to screen for a substance that is effective in treating eating disorders, gastric acid secretion disorders, and / or insulin secretion disorders. The screening procedure is not particularly limited.For example, after screening the receptor of the peptide of the present invention using the peptide of the present invention, the peptide of the present invention is used to screen the receptor for the peptide of the present invention. By screening for a compound that inhibits the binding to the receptor (for example, a receptor agonist or antagonist), a substance effective for treating an eating disorder can be obtained. Hereinafter, screening of the receptor for the peptide of the present invention using the peptide of the present invention (hereinafter referred to as receptor screening) will be described. Subsequently, the present invention will be described using the obtained receptor for the peptide of the present invention. Screening for a compound that inhibits the binding of a peptide to the receptor (hereinafter, referred to as ligand screening) will be described.

Use of the peptide of the present invention enables identification of cells expressing the receptor of the peptide of the present invention, or isolation of the receptor for the peptide of the present invention. When performing screening for isolating the receptor of the peptide of the present invention, the test sample may be, for example, a cell extract of a cell in which the receptor is expected to be expressed, or prepared from the cell. It is possible to use a cDNA expression library prepared based on RNA. NPY family molecules are known to bind to G protein-coupled receptors (GPCRs) as their receptors. Similarly, it is highly likely that the peptide of the present invention also binds to GPCR and performs signal transduction into cells. If the receptor of the peptide of the present invention can be isolated, it is also possible to isolate candidate compounds of the agonist or antagonist relating to the receptor of the peptide of the present invention. Receptor screening can be performed, for example, by the following procedure. First, the purified product is obtained by expressing the peptide of the present invention by genetic recombination technology or by chemical synthesis. The purified peptide was then labeled. And perform binding assays on various cell lines or primary cultured cells, and select cells that express the receptor. [Honjo, Arai, Taniguchi 'Muramatsu, Ed., "New Life Chemistry Experiment Course 7 Proliferation and Differentiation" Factors and Their Receptors "p 203—236 (1991) Tokyo Chemical Dojin. The label, for example, RI labeled (e.g., ³ H or ¹²⁵ 1), or may use an enzyme-labeled (e.g., alkaline phosphatase, etc.). Instead of labeling the peptide of the present invention, an antibody against the peptide of the present invention may be labeled, and the binding between the peptide of the present invention and the receptor may be detected using the labeled antibody.

When the receptor of the present invention or a receptor-expressing cell is obtained by the receptor screening, the binding is inhibited based on the binding activity between the receptor or the receptor-expressing cell and the present peptide. Compounds (eg, receptor agonists and antagonists) can be screened (ligand screening). The test substance to be subjected to this ligand screening method is not particularly limited, and examples thereof include various commercially available compounds, various known compounds registered in a chemical file, peptides, and combinatorial chemistry. A group of compounds obtained by the technique (Terrett, NK et al., Tetrahedron, 51, 8135—8137, 1995), a phage 'display method (FeIici, F. et al., J. Mo. Biol., 222, 301-310, 199 1)), a random peptide group, a culture supernatant of a microorganism, a natural component derived from a plant or marine organism, an animal tissue extract, Alternatively, a compound or peptide obtained by chemically or biologically modifying a peptide can be used.

The substance which affects the activity of the peptide of the present invention is a substance having an agonist activity of the peptide of the present invention (that is, a substance having an activity equivalent to the activity of the peptide of the present invention) by measuring the change in the activity of the peptide of the present invention. Or a substance that enhances the activity) and a substance having an antagonistic activity of the peptide of the present invention (that is, a substance that inhibits the activity of the peptide of the present invention). In this screening method, either a substance having the agonist activity of the peptide of the present invention or a substance having the antagonist activity of the peptide of the present invention can be selected. The screening method of the present invention is more suitable for selecting a substance having an antagonist activity of the peptide of the present invention. In addition, as a primary screening, a substance that binds to the peptide of the present invention is screened, and in a secondary screening, a change in receptor activity of the peptide of the present invention is measured, and screening can be performed by distinguishing agonists or antagonists. Example

Hereinafter, the present invention will be described specifically with reference to Examples, but these do not limit the scope of the present invention.

Example 1: Isolation of a gene encoding a novel NP Y-like peptide of the present invention

The full-length cDNA encoding the peptide of the present invention consisting of the amino acid sequence represented by SEQ ID NO: 2 is obtained by converting a commercially available cDNA derived from human testis (Marathon Readyc DNA; Clontech) into a 錶 type c DNA was obtained by the polymerase chain reaction (PCR) according to the following procedure.

An oligonucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 4 and an oligonucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 5 were used as the first primer and the reverse primer for the first PCR, respectively. did. The first PCR was performed using a DNA polymerase (Pyrobest DNA poIymerase; Takara Shuzo) in the presence of 5% dimethyl sulfoxide (DMSO) at 94 ° C (1 minute) followed by a 94 ° C denaturation step. (30 seconds), 60 ° C (30 seconds) and 72 ° C (1 minute) were repeated 35 times. Subsequently, the reaction solution obtained by the PCR was diluted 50-fold, and a second PCR was performed using the diluted solution as a template. In the second PCR, an oligonucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 6 and an oligonucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 7 were used as the forward primer and the reverse primer, respectively. The PCR was performed under the same conditions as in the first PCR, except that the number was set to 30 times.

As a result of the second PCR, a DNA fragment of about 0.2 kbp was amplified. This fragment was cloned using the pCR2.1 plasmid (Invitrogen). The nucleotide sequence of the obtained clone was analyzed by the dideoxy terminator method using a DNA sequencer (AB I 3700 DNA Sequencer; Ap pl Analysis using ied Biosystems) yielded the nucleotide sequence represented by SEQ ID NO: 1.

The base sequence represented by SEQ ID NO: 1 contained an open reading frame of 213 bases (a sequence consisting of the 36th to 248th bases in the base sequence represented by SEQ ID NO: 1). The amino acid sequence (70 amino acids) predicted from this open reading frame was the amino acid sequence represented by SEQ ID NO: 2. Based on the predicted amino acid sequence obtained, the signal peptide sequence was predicted by the method of von HeiijnΘG (NucIeic Acids Res., 14, 4683–90, 1986). It was found that there was a signal peptide cleavage site between the amino acid (threonine) and the 29th amino acid (cysteine). This proved that this gene was a gene encoding a secretory peptide.

Example 2: BLAST search for SWI SS-PROT in the amino acid sequence of the novel NPY-like peptide of the present invention

For the amino acid sequence of peptide J consisting of the amino acid sequence represented by SEQ ID NO: 2 obtained in Example 1, a BLAST (basic local analysis search) database was searched against the database SWISS-PROT. No sequence identical to the amino acid sequence represented by SEQ ID NO: 2 exists in the peptide, and the peptide YY (PYY) precursor (SWI SS—PROT

56% showed the highest homology (i.e., the number of amino acid residues = 97) to Acc No. P10082 (ideneties). From this, it was revealed that the peptide of the present invention comprising the amino acid sequence represented by SEQ ID NO: 2 was a novel peptide having high homology to PYY belonging to the NPY family.

Example 3: Alignment analysis of the amino acid sequence of the novel NPY-like peptide of the present invention with respect to NPY, PYY, and 属する belonging to known NPY families

Regarding the amino acid sequence of peptide J consisting of the amino acid sequence represented by SEQ ID NO: 2 obtained in Example 1, three types of known peptides belonging to the NPY family, namely, human NPY (GβnBan KA c c. No. XP 004941), human P YY (GenBan K Ac c. No. D1 3899), and human PP (GenBan K Ac c. No. XM 008357). Homology for each amino acid sequence In order to analyze this, an alignment analysis was performed using commercially available software (DNAS IS for Windows Vβr2.1; Hitachi Software Engineering).

The results are shown in Figure 1. In FIG. 1, “XP” represents a peptide consisting of the amino acid sequence represented by SEQ ID NO: 2. Also, "one" indicates a gap portion (that is, no corresponding amino acid is present). As a result of the alignment analysis, the peptide of the present invention consisting of the amino acid sequence represented by SEQ ID NO: 2 is a novel peptide belonging to a novel family that has high homology to a known peptide belonging to the family. In particular, it was found that the homology to 高い最も was the highest. Example 4: BLAST search against human genome draft sequence database with cDNA of novel Ν-like peptide of the present invention

For the cDNA sequence obtained in Example 1, a BLAST search was performed on a human genome draft sequence database. As a result, as a known base sequence including the entire base sequence represented by SEQ ID NO: 1 as a part of the base sequence, GenBank K

No.AL 590366 (chromosome λ clone RP 13-377 G 1, DB: 200 1/05/1 9 P has β 1 Length = 1 94799) and Gen B an KA c c No. AJ 239323 (chrosomesome X clone PACRPCI — 35 19 N 18 map X p 1 1.2, DB: 200 1/05/1 9 P has Θ 2 L engt = 102853) There was. From these results, the “peptide consisting of the amino acid sequence represented by SEQ ID NO: 2” is a peptide whose gene is encoded on the X chromosome, and N PY present on chromosome 7 and PYY present on chromosome 17 And PP were found to be genetically independent on the genome.

Example 5: Confirmation of expression distribution of the gene of the present invention in human tissues

To analyze the tissue expression distribution of the gene encoding peptide J consisting of the amino acid sequence represented by SEQ ID NO: 2 obtained in Example 1, It carried out by the following procedures.

An oligonucleotide consisting of the base sequence represented by SEQ ID NO: 4 and an oligonucleotide consisting of the base sequence represented by SEQ ID NO: 5 as a first primer and a reverse primer for the first PCR, respectively. It was used. First PC R was purified using DNA polymerase (ExTaq DNA polymerase, Takara Shuzo) in the presence of 5% DMSO at 94 ° C (1 minute) followed by 94 ° C (30 minutes). The cycle was composed of 60 ° C (30 seconds) and 72 ° C (45 seconds) for 40 times.

Subsequently, each reaction solution obtained by the PCR was diluted 50-fold, and a second PCR was performed using each of the diluted solutions as a 錶. In the second PCR, an oligonucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 6 and an oligonucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 7 were used as the forward primer and the reverse primer, respectively, and The PCR was performed under the same conditions as the first PCR, except that the number of cycles was changed to 35.

As a result, it was confirmed that a DNA fragment of about 0.2 kbp was amplified by cDNA derived from the hypothalamus, which controls the feeding function. This result supported the feeding function of the peptide encoded by the gene obtained in Example 1. In addition to the hypothalamus, as with PYY and PP, it was confirmed that the DNA was also amplified by each of the pituitary gland and the small intestine-derived cDNA. Therefore, like PYY and PP, they may have both functions of suppressing gastric acid secretion and inhibiting insulin secretion.

Example 6: Construction of FLAG epitope-added XP expression system in animal cells

In this example, the gene encoding the “peptide consisting of the amino acid sequence represented by SEQ ID NO: 2” (hereinafter referred to as XP gene) obtained in Example 1 was referred to as a marker sequence FLAG epitope. An expression vector for expression in animal cells was constructed as a fusion peptide (hereinafter, referred to as XP-FLAG peptide), and the expression of the XP-FLAG peptide was confirmed in animal cells.

Specifically, the amplification of the XP gene (XP-FLAG), which is expressed by in-frame fusion of the marker sequence FLAG epitope at the C-terminal side, is carried out using the nucleotide sequence represented by SEQ ID NO: 8 as a forward primer. And an oligonucleotide consisting of the base sequence represented by SEQ ID NO: 9 was used as a reverse primer. The base sequence represented by SEQ ID NO: 9 includes the FLAG sequence. Thereby, the detection of the expression of the XP peptide can be simplified. In addition, a Kpn I recognition sequence or a Not I recognition sequence is added to the 5 'end of each of the above primers. PCR was carried out at 94 ° C. (for 2 minutes) using DNA probe (Pyrobest DNA polymerase, Takara Shuzo) using the cDNA encoding the XP peptide obtained in Example 1 as type II. After the denaturation step, the cycle consisting of 94 ° C (30 seconds), 55 ° C (30 seconds) and 72 ° C (30 seconds) was repeated 20 times. As a result, a DNA fragment of about 250 bp was amplified. This fragment was cloned using the pCR2.1 plasmid (Invitrogen). The nucleotide sequence of the obtained clone was analyzed by a DNA sequencer (AB13700 DNA Sequencer; Applied Biosystems) using the dideoxy terminator method, and a clone matching the XP-FLAG gene was selected. . The clone was digested with restriction enzymes KpnI and NotI, and inserted into PCEP4 plasmid (InVitrogen) for animal cell expression.

6 wells (Col I agen—Type I—Coated 6 we II p I at Θ; AS AHI TECHNO G LASS) were used for HEK293 cells (1 × 10 ⁵ cells Z ゥ; Invitrogen) ) Was inoculated and cultured for 24 hours, and then the previously constructed XP-FLAG expression plasmid was transfected using a commercially available transfection reagent (FuGENE6; Boeringer Mannheim). 72 hours after the transfection, transfected cells were selected in a hygromycin B-containing medium, and the obtained drug-resistant cells were used as XP-FLAG-expressing cells. After culturing the XP-FLAG expressing cells in a 10 cm Petri dish (Col I agen—Type I—Coated; ASAHI TECHNO G LASS) until confluent, discard the culture medium, After culturing for 4 days in a DMEM medium supplemented with% ゥ fetal serum (FBS), an XP-FLAG peptide-containing medium was recovered.

XP— FLAG peptide expression should be confirmed by Western blot analysis. Specifically, a recovery medium prepared as a sample using SDS (sodi um dodecylsulfate; sodium tridecyl sulfate) Laemmli buffer solution (Daiichi Pure Chemicals) was used for SDSZ1 5% to 25% acrylamide gel. After performing SDS-polyacrylamide gel electrophoresis (SDS-PAGE) using (Daiichi Pure Chemicals Co., Ltd.), it was transferred to a dinitrocellulose membrane. After blocking the obtained transfer membrane with a commercially available blocking reagent (Block Ace: Dainippon Pharmaceutical), A mouse anti-FLAG monoclonal antibody M2 (Sigma) and a horseradish peroxidase-labeled ゥ sagi anti-mouse IgG polyclonal antibody (Zymed) were sequentially reacted. After the reaction, the expression of XP-FLAG peptide was confirmed using a commercially available detection reagent (ECL Western Blotting Detection System; Amersham Pharmacia). The peptide reacting with the anti-FLAG monoclonal antibody M2 is not present in the cells transfected with the empty vector PCEP4, but is detected as a band of about 6 kDa in the medium expressing XP-FLAG peptide. Was. The estimated molecular weight of the XP-FLAG peptide was 61 91.41 Da, with a band at approximately the expected molecular weight.

Example 7: Horizontal construction of screening system for endogenous receptor for XP peptide

In this example, a screening system for an endogenous receptor for XP peptide was constructed.

Specifically, 48 © El plates (Co llage nT ypel -Co ated 48 we II plate; ASAH I TECHNO GLASS Co., Ltd.) in HEK293 cells (3 x 1 0 ⁴ cells well) after seeding and culturing for 24 hours And an orphan receptor expression plasmid (5 ng Zell) and a luciferase 'reporter plasmid pSRE-Iuc or pCRE-Iuc (10 ng / well; manufactured by Stratagen Inc.). The gene was transfected using the Exion reagent (Fu GENE 6). On the next day, the medium was replaced with a medium containing XP-FLAG peptide, and after 5 hours of reaction, the intracellular luciferase activity was measured using a luminometer (ML3000 Iuminometer; Dynatechlaboratories). did.

In this system, the endogenous receptor for XP peptide can be screened by introducing various expression plasmids containing various known orphan receptor genes as the orphan receptor expression plasmid. Industrial applicability

The peptide of the present invention, a polynucleotide encoding the same, an expression vector containing the polynucleotide, and a transformant containing the polynucleotide are used as therapeutic agents for eating disorders, gastric acid secretion disorders, and / or insulin secretion disorders. Useful for manufacturing, It is also useful for screening for a substance that is effective as a therapeutic agent for eating disorders, gastric acid secretion disorders, and / or insulin secretion disorders. Sequence listing free text

The description of “Artificial Sequenc ej” is described in the number heading <223> of the following sequence listing. Specifically, each nucleotide sequence represented by the sequence numbers 8 and 9 in the sequence listing is an artificial sequence. As described above, the present invention has been described in accordance with specific embodiments, but modifications and improvements obvious to those skilled in the art are included in the scope of the present invention.

Claims

The scope of the claims

1. A peptide consisting of the amino acid sequence represented by SEQ ID NO: 3.

2. A peptide consisting of the amino acid sequence represented by SEQ ID NO: 2.

3. A polynucleotide encoding the peptide of claim 1 or 2.

4. An expression vector comprising the polynucleotide of claim 3.

5. A transformant comprising the polynucleotide according to claim 3.

6. The method for producing a peptide according to claim 1 or 2, comprising a step of culturing the transformant according to claim 5, and a step of recovering the peptide.

7. A peptide that can be produced by a method comprising a step of culturing a transformed eukaryotic cell containing a polynucleotide encoding a peptide consisting of the amino acid sequence represented by SEQ ID NO: 2, and a step of recovering the peptide.

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