CN1253178A - Protein correlative to obesity inhibin receptor, coding sequence and its preparing process and application - Google Patents

Abstract

The present invention discloses an obesity inhibin receptor correlative protein OB-RGRP2 and its nucleic acid coding sequence. Said protein is high homogeneous with OB-RGRP. The process for preparing said nucleic acid sequence and protein polypeptide, and the application of said polypeptide and nucleic acid sequence are also disclosed.

Description

New protein correlative to obesity inhibin receptor and encoding sequence, and method for making and purposes

The invention belongs to biotechnology and genetically engineered field, more particularly, the present invention relates to a kind of new protein correlative to obesity inhibin receptor OB-RGRP2 and nucleic acid coding sequence thereof, the method for producing this nucleotide sequence and protein polypeptide, and the purposes of this polypeptide and nucleotide sequence.

Obesity is a modal nutritive disease in the western developed country.Grownup's body weight more than 30% 20% of body weight that is above standard.Therefore, obesity becomes important public health problem.In addition, fat normal and diabetes, essential hypertension, hyperlipidaemia, cardiovascular and cerebrovascular disease even cancer are closely related.Thereby people have dropped into great amount of manpower and material resources to the research of obesity always.Yet fat molecular mechanism is unclear always.

For many years it is believed that fat relevant with gene with heredity.In recent years from a kind of mouse that suffers from obesity, be cloned into one with fat closely-related gene-ob gene, one of this genes encoding is regulated the body energy equilibrated hormones factor, be called obesity inhibin (leptin, also the someone is translated into the element of becoming thin), afterwards, in the mankind, also be cloned into homologous OB gene [Zhang Y, et al.Nature 1994; 372:425].Human OB gene is located in 7q31.3.The former albumen of obesity inhibin that the OB genes encoding is made up of 167 amino-acid residues, after the signal peptide of 21 amino acid longs having removed the N-end, the ripe obesity inhibin of formation contains 146 amino acid.Studies show that obesity inhibin is mainly secreted by the fat tissue cell of white.

Research shows that also obesity inhibin has many-sided biological action [Halaas JL, et al.Science1995; 269:543]:

(1) balance of adjusting energy metabolism.This is one of most important function of obesity inhibin, mainly regulates the absorption of food and the consumption of stimulation energy (comprise and generate heat).Its site of action and approach have two:

A. act on the nervus centralis endocrine system: main target site is a hypothalamus.Obesity inhibin enters central nervous system through the infiltration transporting mode by hemato encephalic barrier.Obesity inhibin downstream effects molecule may be hypothalamic neuropeptide Y (NPY).NPY can stimulate the absorption of food, and that obesity inhibin can suppress NPY is synthetic.In addition, some other factor or hormone such as thyroliberin (ACTH), glycogen sample polypeptide etc. also may be relevant with the activity of obesity inhibin.

B. act on peripheral tissues: obesity inhibin is brought into play the effect of regulating energy balance by the regulation and control lipid metabolism.Obesity inhibin can directly suppress the synthetic of lipid acid and triglyceride level in the cell, increases the oxidation of lipid simultaneously, thereby causes that the content of lipid acid and triglyceride level descends in the cell.Studies show that further the increase of oxygenizement and increase without the ATP synthetic in the plastosome that is to say that the energy that oxygenizement increased has been converted into heat mostly.

(2) obesity inhibin also has important regulatory role to other system.For example A. is to reproductive system, and obesity inhibin plays an important role to embryo's growth and maturation.B. to hemopoietic system, obesity inhibin can directly act on early stage hematopoiesis and stimulate red system and grain is the growth and the differentiation of progenitor cell.This prompting, obesity inhibin has therapeutic value to hemopathy, separates the early stage hemopoietic progenitor cell of OB-R male such as can be used for, and this is significant to hematopoietic stem cell transplantation.Also can be used for treating anaemia in addition and strengthen macrophage function and immunizing power.

Obesity inhibin is to work by the mediation of the obesity inhibin receptor that is positioned at cell surface (being called for short " OB-R ").OB-R is a kind of transmembrane protein, and there is quite high homology in the acceptor gp130 subunit of it and interleukin-6, belongs to cytokine I class family [Tartaglia LA, et a1.Cell 1995; 83:1263].

Found to mainly contain two types acceptor: OB-RL and OB-RS.OB-RL is made up of more than 3000 amino-acid residues, and part is about 303 amino acid in its born of the same parents, contain and the relevant structural domain of intracellular signal conduction, as with the motif of Jak bonded motif (Motif) and activation STAT.OB-RS has lacked the STAT motif, but its born of the same parents' outside part and OB-RL are identical.Have four kinds of OB-RS that are uneven in length at present at least.

Another kind of shorter obesity inhibin receptor has also lacked strides membrane portions, and it is a kind of soluble components that is present in the blood, might be the conjugated protein of a kind of obesity inhibin.Its source may be that to enter blood by OB-RS born of the same parents' outside part after the proteolytic enzyme effect formed.

OB-RL mainly expresses in hypothalamus, and expression amount is few in its hetero-organization.And OB-RS all has expression in various degree in various tissues, and is the highest to express in the tissues such as lung and kidney.The structure of two kinds of OB-R and the difference of express spectra have determined on both functions different.Think that at present the effect of obesity inhibin is mainly mediated by OB-RL.

A kind of cause of disease of obesity is owing to chalone biosynthesis block or the vigor forfeiture of causeing fat of obesity inhibin genetic flaw, but the obesity sickness rate due to this obesity inhibin genetic flaw is extremely low.Most of obesity may be resisted obesity inhibin due to (leptin resistance).Think that now the obesity inhibin opposing mainly contains two kinds of reasons: the A. obesity inhibin enters hypothalamic process by blood by hemato encephalic barrier and is obstructed, thereby make fatty tissue excretory obesity inhibin can not pass through hemato encephalic barrier effectively, yet central nervous system is still responsive to obesity inhibin.Can infer to treating this obesity important value to be arranged by free OB-R activator thus by hemato encephalic barrier.B. factor generation function reduction or the forfeiture behind obesity inhibin receptor or the acceptor, thus cause resisting phenomenon.All may fall ill relevant with the function reduction or the forfeiture of arbitrary link in obesity inhibin receptor and the downstream signal pathway with obesity.As seen, OB-R is one of important step of obesity morbidity, also is simultaneously important target site [the Tartaglia LA.J Bio Chem 1997 of treatment and obesity prevention; 272:6093].

Recently, there is the scholar to be cloned into a gene with the OB-R gene-correlation.This gene contains the open reading frame (ORF) of a 396bp, 131 the amino acid whose protein of encoding, called after OB-RGRP (OB-R Gene relatedprotein is translated into " obesity inhibin receptor gene-correlation albumen ").The sequence (164bp) of preceding 2 exons of OB-RGRP is with 5 of OB-R '-the non-translational region sequence homology is 100%, but later sequence different fully [Bailleul B, the et al.Nucleic Acids Res 1997 with OB-R of 165bp; 25:2752].Therefore the author proposes, and OB-RGRP and OB-R belong to same locus, are controlled by same promotor, form different transcription products through different exon montage processes.

OB-RGRP is carried out the homology comparative analysis, find the higher homology of C30B5.2 albumen with nematode.Two structural domains (domain) height homology is arranged: the structural domain 1 that (1) is from+9 to+27, this structural domain is made up of highly hydrophobic and nonpolar amino acid; (2) from+65 to+88 structural domain 2.These structural domains may be to stride membrane structure.In many cytokine receptors (comprising OB-R) family, have after a Pro-X-Pro sequence (in the formula, X represents arbitrary amino-acid residue) is positioned at one group of hydrophobic residue at the near-end of striding membrane structure, this sequence is called box1.If two Pro are substituted by Ser, then receptor-mediated JAK2 tyrosine phosphorylation disappears.In OB-RGRP, also there is a similar box1 (Pro ₄₆-Ile-Pro ₄₈).Known at present, OB-RL can guide the JAK/STAT signal transduction path, but OB-RS then can not guide this pathway.And two kinds of OB-R contain the box1 sequence, so this explanation box1 still is not enough to activate the JAK/STAT system.But two kinds of OB-R can both induce the downstream signaling molecule expression of gene under the stimulation of obesity inhibin, and this prompting OB-RGRP may be important collaborative albumen in the obesity inhibin signal conductive process, and especially the signal to the OB-RS mediation conducts.

Because OB-R belongs to I type cytokines receptor family, with the OB-R 26S Proteasome Structure and Function mutually Sihe signal transduction path other I type cytokines such as Interferon, rabbit, interleukin-6 receptor etc. roughly the same similarly collaborative albumen may also be arranged.

But, before the application, do not deliver or disclosed new protein correlative to obesity inhibin receptor and encoding sequence involved in the present invention.

First purpose of the present invention provides a kind of new nucleic acid coding sequence, a newcomer of this nucleic acid sequence encoding protein correlative to obesity inhibin receptor family, and the new people's gene of the present invention is named as OB-RGRP2.

Second purpose of the present invention provides a kind of new protein correlative to obesity inhibin receptor, and this albumen is named as OB-RGRP2 albumen.

The 3rd purpose of the present invention provides utilizes recombinant technology to produce the described new people OB-RGRP2 nucleotide sequence and the method for polypeptide.

The 4th purpose of the present invention provides the application of people OB-RGRP2 nucleotide sequence and polypeptide.

The 5th purpose of the present invention provides the antibody at OB-RGRP2.

In one aspect of the invention, a kind of isolated nucleic acid molecule is provided, it comprises: coding has the nucleotide sequence of the polypeptide of people OB-RGRP2 protein-active, and the nucleotide sequence of 85-480 position has 70% homology at least among this nucleotide sequence and the SEQ ID No.1; Perhaps this nucleotide sequence can be under the height stringent condition with SEQ ID No.1 in the nucleotide sequence hybridization of 85-480 position.Preferably, this nucleotide sequence coded polypeptide with aminoacid sequence shown in the SEQ ID No.2.More preferably, this nucleotide sequence has the sequence of 85-480 position among the SEQ ID No.1.

In another aspect of this invention, provide a kind of isolating OB-RGRP2 polypeptide, it comprises: polypeptide or its conservative property variation polypeptide or its active fragments or its reactive derivative with SEQID No.2 aminoacid sequence.Preferably, this polypeptide has SEQ ID No.2 aminoacid sequence.

In another aspect of this invention, provide the carrier that contains above-mentioned nucleic acid molecule.

In another aspect of this invention, provide with described carrier transformed host cells.

In another aspect of this invention, provide the preparation method of the polypeptide with people OB-RGRP2 protein-active, this method comprises:

(a) nucleotide sequence that coding is had a polypeptide of OB-RGRP2 protein-active operationally is connected in expression regulation sequence, form the OB-RGRP2 protein expression vector, the sequence from Nucleotide 85-480 position among described nucleotide sequence and the SEQ ID NO.1 has at least 70% homology;

(b) change the expression vector in the step (a) over to host cell, form the proteic reconstitution cell of OB-RGRP2;

(c) be fit to express under the condition of OB-RGRP2 protein polypeptide the reconstitution cell in the culturing step (b);

(d) isolate polypeptide with OB-RGRP2 protein-active.

In another aspect of this invention, provide OB-RGRP2 polypeptid specificity bonded antibody that can be above-mentioned

The new albumen relevant with obesity inhibin receptor provided by the present invention is named as OB-RGRP2.On the one hand, OB-RGRP2 can promote the signal conduction of obesity inhibin as the collaborative albumen of obesity inhibin (OB-R); OB-RGRP2 has the effect of collaborative other I type cytokines acceptor on the other hand, at aspects such as cells whose development and differentiation, metabolism, adjusting immunologic functions critical function is arranged all.The dysfunction of OB-RGRP2 can cause disorder, malignant tumour of disease, diabetes, the immunologic function of obesity, hemopoietic system etc.The disease that the antibody of anti-OB-RGRP2 can be used for diagnosing or treatment is relevant with OB-RGRP2.

One of feature of the present invention is a kind of nucleic acid (polynucleotide) sequence of isolating or purifying, and this nucleic acid sequence encoding has the OB-RGRP2 polypeptide of its acid sequence of ammonia shown in the SEQ ID NO:2.Because the degeneracy of codon, thus with SEQ ID NO.1 in 85-480 bit sequence homology be low to moderate about 70% the degenerate sequence described aminoacid sequence of SEQ ID NO.2 of also encoding out.Preferably, this nucleotide sequence is substantially the same with the sequence shown in the SEQ ID NO:1.The present invention's said " substantially the same " is meant homology at least 80%, preferably at least 90%, better at least 95%, preferably at least 99%.

Polynucleotide of the present invention can be dna form or rna form.Dna form comprises cDNA, genomic dna, or the DNA of synthetic.DNA can be strand or two strands.The DNA of strand can be coding strand or noncoding strand.The coding region sequence of encoding mature polypeptide can with the identical or degeneracy varient of coding region sequence (85-480bp) shown in the SEQ ID NO:1.In the present invention, " degeneracy varient " be meant that coding has the protein of SEQ ID NO:2 sequence or peptide but with the differentiated nucleotide sequence of coding region sequence shown in the SEQ ID NO:1.

The present invention also comprised isolated or purified, under stringent condition can with the nucleic acid fragment of SEQ ID NO:1 sequence (especially open reading frame sequence) hybridization.Nucleic acid fragment in the present invention, " " length contain 10 Nucleotide at least, preferably at least 20 Nucleotide, more preferably at least 40 Nucleotide, at least 60 Nucleotide best." hybridization " is meant with one section special nucleotide sequence and combines.As is known to the person skilled in the art, " stringent condition " typically refers to: hybridization under (1) low ionic strength and the high-temperature and wash-out, and such as 0.2 * SSC, 0.1%SDS, 60 ℃; Or (2) hybridization the time adds and to use denaturing agent, such as 50% (v/v) methane amide, and 0.1% calf serum/O.1%Ficoll, 42 ℃ etc.; Or (3) homogeny at least 95% between two sequences only, just hybridize at least 97% the time better.Nucleic acid fragment can be used for the amplification technique (as PCR) of nucleic acid, so that determine and/or isolate the polynucleotide of coding OB-RGRP2.

The coding region sequence of polynucleotide of the present invention can be the allelic variation body of the coding region sequence shown in SEQ ID NO:1.Prior art shows, the allelic variation body is one or morely in the polynucleotide (to be generally 1-60, more preferably 1-20,1-10 best) Nucleotide substitutes, lacks or insert formed replacement form, several (are generally in 60 also to be included in 5 ' and/or 3 ' end interpolation, preferably being in 30, more preferably is in 10, is in 5 best) the formed replacement form of Nucleotide.These replacement forms do not change the function of coded albumen or polypeptide basically.

In an example of the present invention, the nucleic acid molecule with sequence shown in the SEQ ID No.1 is provided, wherein, open reading frame is positioned at 85-480 position Nucleotide.

The present invention also provides the OB-RGRP2 polypeptide of isolated or purified, such as the polypeptide with SEQ ID NO:2 aminoacid sequence.

In the present invention, term " OB-RGRP2 albumen and/or polypeptide " refers to have the polypeptide of aminoacid sequence shown in the SEQ ID NO.2 of OB-RGRP2 protein-active.This term also comprises having and variant form people OB-RGRP2 albumen identical function, SEQ ID NO.2 sequence.These variant forms comprise (but being not limited to): several (are generally 1-30, more preferably 1-20,1-10 best) amino acid whose disappearance, insertion and/or replacement, and add one or several at C-terminal and/or N-terminal and (be generally in 20, preferably being in 10, more preferably is in 5) amino acid.As well known to the skilled person, when replacing, can not change proteinic function usually with the close or similar amino acid of performance.Again such as, add one or several amino acid at C-terminal and/or N-terminal and also can not change proteinic function usually.This term also comprises proteic active fragments of OB-RGRP2 and reactive derivative.

The variant form of this polypeptide comprises: homologous sequence, allelic variant, natural mutation, induced mutation body, under high or low rigorous degree condition can with the coded albumen of the DNA of OB-RGRP2 DNA hybridization and the polypeptide or the albumen that utilize the antiserum(antisera) of anti-OB-RGRP2 polypeptide to obtain.The present invention also provides other polypeptide, as comprises OB-RGRP2 polypeptide or its segmental fusion rotein.Except the polypeptide of total length almost, the present invention also provides the soluble fragments of OB-RGRP2 polypeptide.Usually, this fragment have the OB-RGRP2 peptide sequence at least about 10 continuous amino acids, preferably at least about 30 continuous amino acids, more preferably at least about 80 continuous amino acids, best at least about 100 continuous amino acids.

Invention also provides the analogue of OB-RGRP2 albumen or polypeptide.The difference of these analogues and natural OB-RGRP2 polypeptide can be the difference on the aminoacid sequence, also can be the difference that does not influence on the modified forms of sequence, perhaps haves both at the same time.These polypeptide comprise natural or the inductive genetic variant.The induce variation body can obtain by various technology, as passing through site-directed mutagenesis method or the biological technology of other known moleculars.Analogue can also comprise having the analogue that is different from the amino acid whose residue of natural L-(as D-amino acid), and has non-natural analogue that exist or synthetic amino acid (as β, gamma-amino acid).

(the not changing primary structure usually) form of modification comprises: the chemically derived form such as the acetylize or carboxylated of the polypeptide that body is interior or external.Modification also comprises glycosylation, carries out glycosylation modified and polypeptide that produce in the procedure of processing as those in the synthetic and processing of polypeptide or further.This modification can be carried out glycosylated enzyme (as mammiferous glycosylase or deglycosylating enzyme) and finishes by polypeptide is exposed to.

In the present invention, " OB-RGRP2 conservative property variation polypeptide " refers to compare with the aminoacid sequence of SEQ ID No.2, has 10 at the most, and preferably at the most 8, more preferably 5 amino acid is replaced by similar performance or close amino acid and formed polypeptide at the most.These conservative property variation polypeptide are preferably replaced according to table 1 and are produced.

Table 1

Initial residue	Representational replacement	The preferred replacement
			Ala(A)	Val；Leu；Ile	Val
Arg(R)	Lys；Gln；Asn	Lys
			Asn(N)	Gln；His；Lys；Arg	Gln
Asp(D)	Glu	Glu
			Cys(C)	Ser	Ser
Gln(Q)	Asn	Asn
			Glu(E)	Asp	Asp
Gly(G)	Pro；Ala	Ala
			His(H)	Asn；Gln；Lys；Arg	Arg
Ile(I)	Leu；Val；Met；Ala；Phe	Leu
			Leu(L)	Ile；Val；Met；Ala；Phe	Ile
Lys(K)	Arg；Gln；Asn	Arg
			Met(M)	Leu；Phe；Ile	Leu
Phe(F)	Leu；Val；Ile；Ala；Tyr	Leu
			Pro(P)	Ala	Ala
Ser(S)	Thr	Thr
			Thr(T)	Ser	Ser
Trp(W)	Tyr；Phe	Tyr
			Tyr(Y)	Trp；Phe；Thr；Ser	Phe
Val(V)	Ile；Leu；Met；Phe；Ala；	Leu

In the present invention, " isolating or purifying " is meant material be separated (if crude substance, primal environment is exactly a natural surroundings) from its primal environment.For example, in active somatic cell, be in polynucleotide and polypeptide under the native state and be and do not separate with purifying, if but separate in simultaneous other materials in same polynucleotide or polypeptide and the native state, be exactly isolated or purified.

The present invention also comprises nucleic acid molecule or its varient or its segmental carrier that contains open reading frame sequence among the SEQ ID NO:1, also comprise with this carrier and carry out genetically engineered host cell, and the albumen of the present invention or the polypeptide product that obtain with the DNA recombinant technology.

The present invention also provides the method for the diagnosis disease relevant with the OB-RGRP2 dysfunction, detects the expression of OB-RGRP2 in tissue or the cell or the sudden change of OB-RGRP2 gene comprising (but being not limited to).By the mRNA level of measuring OB-RGRP2 in tissue or the cell or the expression that protein level can be judged OB-RGRP2.

The present invention also provides the method for the treatment disease relevant with the OB-RGRP2 dysfunction, uses the mixture of significant quantity comprising (but being not limited to) to cell, with generation or the activity of activation or inhibition OB-RGRP2.The kind of this mixture comprises: compound; With OB-RGRP2 bonded part, polypeptide or antibody; Antisense oligonucleotide; Ribozyme (Ribozyme); And their mixture.

The method of cloning OB-RGRP2 gene of the present invention is that the skilled person uses always in this area.A kind of feasible approach is to be used to come from the mRNA of mammalian tissues (as embryo and brain or liver or lung etc.) cell through the synthetic cDNA of reverse transcription mode, and makes up corresponding cDNA library, obtains to contain the clone of OB-RGRP2 gene again from the library.

Extract the existing multiple proven technique of method of mRNA, relevant test kit also can obtain from commercial channels.The method in construction cDNA library also is conventional method (Sambrook, et al, Molecular Cloning, aLaboratory Manual, Cold Spring Harbor Laboratory.New York, 1989).In addition also directly from commercial channels (as Clontech) obtain the cDNA library of multiple tissue.

From the cDNA library, obtain the OB-RGRP2 gene, include, but is not limited to following two kinds of technological lines: the one, OB-RGRP2 cDNA clone is contained in the screening library earlier, again these positive colonies is carried out dna sequence analysis; The 2nd, measure earlier 5 ' and 3 ' sequence of the insertion dna fragmentation of all clones in the library, and then find out the clone who contains OB-RGRP2 cDNA, and carry out the sequential analysis of full-length cDNA.

The method in screening library includes, but is not limited to: (1) DNA-DNA or DNA-RNA hybridization; (2) function of marker gene manifests or loses; (3) level of the transcript of mensuration OB-RGRP2; (4) applied immunology technology or mensuration biologic activity detect the protein product of genetic expression.Aforesaid method can use separately, but also several different methods applied in any combination.

In (1) kind method, it is identical with any part of OB-RGRP2 dna fragmentation (fragment of sequence shown in SEQ ID No.1) hybridizing used probe, at least 10 continuous nucleotides of the length of probe, better be at least about 20-30 continuous nucleotide, be more preferably at least about 50-60 continuous nucleotide, preferably at least about 100 Nucleotide.The length of probe is generally in the 5kb, preferably in the 2kb, more preferably in the 1kb, best in the 500bp.The mark of dna probe can be used radio isotope, fluorescein or enzyme (as alkaline phosphatase) etc.

In (4) kind method, the protein product that detects OB-RGRP2 genetic expression can carry out with immunological technique such as Western blotting, radioimmunoprecipitation, enzyme-linked immunosorbent assay (ELISA) etc.

The dideoxy chain termination of the available routine of mensuration of dna sequence dna (Sanger et al.PNAS, 1977,74:5463-5467), and the used primer that checks order can known cDNA sequence (as the sequence of SEQ ID No.1) design.

In order to obtain the cDNA sequence of total length, order-checking need be carried out repeatedly.Sometimes need to measure a plurality of clones' cDNA sequence, just can be spliced into the cDNA sequence of total length.

If fail to obtain complete 5 ' end encoding sequence (this is the problem that often runs into) in the cDNA clone, then need to carry out RACE (the terminal rapid amplifying technology of cDNA).

In case obtain the cDNA sequence of total length, then available computers is analyzed sequence, relatively wait as the position of open reading frame, possible signal peptide, the homology of striding membrane structure and gene or protein structure.

In addition, obtaining full length cDNA sequence, just can obtain the proteic encoding sequence of the present invention by the pcr amplification method with ordinary method design and synthetic relevant primer.

OB-RGRP2 polymerized nucleoside acid sequence of the present invention can be used to express or produce the OB-RGRP2 albumen or the polypeptide of reorganization.In general following steps are arranged:

(a) nucleotide sequence that coding is had a polypeptide of OB-RGRP2 protein-active operationally is connected in expression regulation sequence, form the OB-RGRP2 protein expression vector, the sequence from Nucleotide 85-480 position among described nucleotide sequence and the SEQ ID NO.1 has at least 70% homology;

(b) change the expression vector in the step (a) over to host cell, form the proteic reconstitution cell of OB-RGRP2;

(c) be fit to express under the condition of OB-RGRP2 protein polypeptide the reconstitution cell in the culturing step (b);

(d) isolate polypeptide with OB-RGRP2 protein-active.

In step (a), the nucleotide sequence that used coding has the polypeptide of OB-RGRP2 protein-active can be dna fragmentation or its varient with SEQ ID No.1 coding region.

" expression regulation sequence " comprises that at least one promotor, at least one terminator codon and other are any and transcribes and the institute of translation subsequently essential or preferred dna sequence dna, for example leader sequence, an enhanser etc. for the suitable of vector nucleic acid sequence.

As used herein, " operationally being connected in " refers to a kind of like this situation, and promptly some part of linear DNA sequence can influence the activity of same other parts of linear DNA sequence.For example, if signal peptide DNA as precursor expression and participate in the secretion of polypeptide, signal peptide (secretion leader sequence) DNA operationally is connected in polypeptid DNA so; If transcribing of promotor control sequence, it is operationally to be connected in encoding sequence so; When if ribosome bind site is placed in the position that can make its translation, it is operationally to be connected in encoding sequence so.Generally, " operationally being connected in " means adjacent, then means in reading frame adjacent for the secretion leader sequence.

A key character of expression vector is to contain suitable promotor and translation controlling elements.Multiple expression vector can obtain from commercial channels.Carrier commonly used includes, but is not limited to: plasmid, phage, clay, Yeast expression carrier, virus, Ti-plasmids etc., but the first-selected plasmid of the most frequently used carrier.Host cell commonly used includes, but is not limited to: bacterium, yeast, insect cell, zooblast or vegetable cell etc., but the most frequently used host cell is a bacterium, especially intestinal bacteria.Another expression system commonly used is a mammal cell line, as Hela clone, COS clone or Chinese hamster ovary celI system etc.

The known method of skilled person makes up the recombinant expression vector that contains the OB-RGRP2 encoding sequence in available this area.These methods comprise: and extracorporeal recombinant DNA technology, DNA synthetic technology, the interior recombinant technology of body etc. (referring to: Sambrook, et al, Molecular Cloning, a Laboratory Manual, Cold Spring HarborLaboratory.New York, 1989).

The OB-RGRP2 albumen or the polypeptide of reorganization are of use in many ways.These purposes include, but is not limited to: directly as the disease due to pharmacological agent OB-RGRP2 hypofunction or the forfeiture be used to screen and promote or antibody, polypeptide or other part of antagonism OB-RGRP2 function.For example, antibody can be used for activating or suppressing the function of OB-RGRP2.In addition, can be used for seeking peptide molecule therapeutic value, that can suppress or stimulate the OB-RGRP2 function with the reorganization OB-RGRP2 protein screening peptide library of expressing.

The present invention also provides can be specifically and OB-RGRP2 bonded antibody or antibody fragment.Antibody fragment has Fab, Fab ', F (ab ') ₂Or Fr fragment.Antibody can be antibody strand, humanized or chimeric.

There is several different methods to can be used for producing antibody at the OB-RGRP2 antigenic determinant.These antibody include, but is not limited to: the fragment that polyclonal antibody, monoclonal antibody, chimeric antibody, single-chain antibody, Fab fragment and Fab expression library produce.

The antibody of anti-OB-RGRP2 can be used for immunohistochemistry technology, with whether existing of the OB-RGRP2 in the detection biopsy sample.With the also available labelled with radioisotope of OB-RGRP2 bonded monoclonal antibody, inject position and the distribution that to follow the tracks of OB-RGRP2 in the body.This radiolabeled antibody can be used as a kind of atraumatic diagnostic method and is used for the location of relevant cell, as judges whether tumour cell has transfer etc.

The disease that antibody among the present invention can be used for treating or prevention is relevant with OB-RGRP2.The antibody that gives suitable dosage can stimulate or block generation or the activity of OB-RGRP2.

Antibody also can be designed to the immunotoxin at a certain privileged sites in the body.As the monoclonal antibody of OB-RGRP2 high-affinity can with bacterium or plant poison (as diphtheria toxin, ricin, abrine etc.) covalent attachment.A kind of usual method is with the amino group on sulfydryl linking agent such as SPDP (N-succinimide-3-(2-pyridine dithio)-propionic ester) the attack antibody, by the exchange interaction of disulfide linkage toxin is incorporated on the antibody.This hybrid antibody can be used for killing the OB-RGRP2 positive cell.

Available OB-RGRP2 albumen of the production of polyclonal antibody or polypeptide immune animal are as rabbit, mouse and rat etc.Multiple adjuvant can be used for enhancing immunity reaction, comprising (but being not limited to) freund's adjuvant etc.

The OB-RGRP2 monoclonal antibody can with hybridoma technology production (Kohler and Milstein.Nature, 1975.256:495-497).With the variable region bonded chimeric antibody in human constant region and inhuman source can with existing technology production (Morrison et al, PNAS, 1985,81:6851).In addition, the technology of existing manufacture order chain antibody (U.S. Patent No. 4946778) also can be used for producing the single-chain antibody of anti-OB-RGRP2.

Can with OB-RGRP2 bonded peptide molecule, can be by screening and obtain to be incorporated into rondom polypeptide storehouse that solid formation forms by the various amino acid that may make up.

The OB-RGRP2 polynucleotide also can be used for the diagnosis and the treatment of OB-RGRP2 relative disease.Aspect diagnosis, whether the expression that the OB-RGRP2 polynucleotide can be used for detecting whether expressing of OB-RGRP2 or OB-RGRP2 unusual (under at morbid state).Can be used for the hybridization of biopsy sample to judge the abnormal expression of OB-RGRP2 as the OB-RGRP2 dna sequence dna.Hybridization technique comprises Southern blotting, Northern blotting and in situ hybridization etc.These technological methods all are disclosed mature technologies, and relevant test kit all can obtain from commercial channels.In addition, carry out the transcription product that RNA-polymerase chain reaction (PCR) amplification in vitro also can detect OB-RGRP2 with the special primer of OB-RGRP2.Wherein specific primer can design according to SEQ ID No.1 sequence, and its length is generally 15-60 Nucleotide, preferably is 20-50 Nucleotide, more preferably is 25-40 Nucleotide.

In addition, the sudden change that detects the OB-RGRP2 gene also can be used for the disease of diagnosing OB-RGRP2 relevant.The form of OB-RGRP2 sudden change comprises: point mutation, transposition, disappearance, reorganization and compare with normal wild type OB-RGRP2 dna sequence dna other are any unusual etc.Available existing technology such as Southern blotting, dna sequence analysis, PCR and in situ hybridization detect sudden change.In addition, sudden change influences proteic expression sometimes, therefore can judge indirectly that with Northern blotting and Western blotting gene has or not sudden change.

The OB-RGRP2 polynucleotide also can be used for multiple therepic use.Gene therapy technology can be used for treating because cell proliferation, growth or the metabolic disturbance due to the expression of the OB-RGRP2 of the nothing expression of OB-RGRP2 or unusual/non-activity.The OB-RGRP2 that the gene therapy vector (as virus vector) of reorganization can be designed to express variation is used to suppress endogenic OB-RGRP2 activity.For example, a kind of OB-RGRP2 of variation can be OB-RGRP2 brachymemma, that lacked signal conduction function territory.Therefore, though the OB-RGRP2 of this variation can combine with the substrate in downstream, lack signaling activity.Like this, the gene therapy vector of reorganization just is used for the treatment of the disease of OB-RGRP2 expression or active caused by abnormal.Deriving from viral expression vector such as retrovirus, adenovirus, adeno-associated virus (AAV), hsv, parvovirus etc. can be used for the OB-RGRP2 transgenosis to cell.The method that structure carries the recombinant viral vector of OB-RGRP2 gene is found in existing document (Sambrook, et al, Molecular Cloning, a Laboratory Manual, Cold Spring HarborLaboratory.New York, 1989).In addition, reorganization OB-RGRP2 gene can be packaged in the liposome and is transferred in the cell.

Suppress the oligonucleotide (comprising sense-rna and DNA) of OB-RGRP2mRNA translation and ribozyme also within the scope of the invention.Ribozyme is the enzyme sample RNA molecule that a kind of energy specificity is decomposed specific RNA, and its mechanism of action is to carry out the endonuclease effect after ribozyme molecule and the hybridization of complementary target RNA-specific.The RNA of antisense and DNA and ribozyme can obtain with the technology of synthetic RNA of existing any routine or DNA, as the technology of the solid phase phosphoamide chemical synthesis synthetic oligonucleotide of widespread use.Antisense rna molecule also can be transcribed acquisition by the dna sequence dna of this RNA that encodes in external or body, wherein this dna sequence dna has been incorporated into the downstream of rna polymerase promoter of carrier.In order to increase the stability of nucleic acid molecule, available several different methods is modified it, for example increases the sequence length of both sides, makes oligonucleotide use the phosphoric acid thioester bond but not phosphodiester bond.

Polynucleotide imports tissue or intracellular method is a various routine techniques as known in the art, comprising (but being not limited to): directly be injected into polynucleotide in the in-vivo tissue; Or external by carrier (as virus, phage or plasmid etc.) earlier with the polynucleotide transfered cell in, again cell is transplanted in the body etc.

In the accompanying drawings, Fig. 1 is the homology comparison diagram of the aminoacid sequence of people OB-RGRP2 of the present invention and OB-RGRP.Wherein, identical amino acid marks with the amino acid monocase between two sequences, and similar amino acid marks with "+".

Fig. 2 is the homology comparison diagram of aminoacid sequence of the C30b5 of people OB-RGRP2 of the present invention and nematode.Wherein, identical amino acid marks with the amino acid monocase between two sequences, and similar amino acid marks with "+".

Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, (such as people such as Sambrook, molecular cloning: laboratory manual (New York:ColdSpring Harbor Laboratory Press, 1989) condition described in), or according to the condition that manufacturer advises carry out.

The clone of embodiment 1:OB-RGRP2 gene

Extract the total RNA of people's tire brain with guanidinium isothiocyanate/phenol/chloroform single stage method.From total RNA, separate poly (A) mRNA with Quik mRNA Isolation Kit (Qiegene).2 microgram poly (A) mRNA form cDNA. through reverse transcription and with UniZAPxR support agent box (available from Stratagene) cDNA fragment orientation are inserted on the multiple clone site of carrier, transform the DH5 bacterium and form the cDNA library.Obtain 3028 clones altogether.Measure all clones' 5 ' and 3 ' terminal sequence with dideoxy method.CDNA sequence and the existing public dna sequence data storehouse measured are compared, found that a clone's (018e11) dna sequence dna and the homology of people OB-RGRP reach 70.2%.By synthetic a series of primers the contained dna sequence dna of 018e11 clone is carried out two-way mensuration.Computer Analysis shows, the contained full-length cDNA of 018e11 clone is a new dna sequence dna (shown in SEQ ID No.1), from 85bp to 480bp the ORF of a 396bp is arranged, the new protein that constitutes by 131 amino acid of encoding, its aminoacid sequence is shown in SEQ ID No.2.This protein is named as OB-RGRP2 albumen.

OB-RGRP2 contains 131 amino acid, and is identical with the length of OB-RGRP.The homology analysis of protein level shows that OB-RGRP2 and OB-RGRP homogeny are 67%, and similarity is 80% (Fig. 1).With the C30b5 albumen homogeny of nematode be 48%, similarity is 78% (Fig. 2).Structural analysis shows that there are two structural domains of mainly being made up of hydrophobic amino acid (9-27 position and 65-88 position) in OB-RGRP2, and they are estimated to be strides membrane structure, thereby OB-RGRP2 is a transmembrane protein.Have a LRRs motif in structural domain 1 district, this motif is rich in leucine, and its function mainly participates in the interaction between the protein-protein.In the downstream of structural domain 1, also there is the motif (46-48 amino acids Pro Ile Pro among the SEQ ID No.2) of a Pro-X-Pro, this motif is relevant with the function of Jak2.The above results shows that OB-RGRP2 and OB-RGRP belong to a gene family, has the similar function of OB-RGRP, promptly promotes the signal conduction of obesity inhibin as the collaborative albumen of OB-R; In addition, also may have the effect of collaborative other cytokine receptor, thereby its function ratio OB-RGRP is more extensive.All play an important role at aspects such as cells whose development and differentiation, metabolism, adjusting immunologic functions.

People OB-RGRP2 of the present invention is used for further functional study except can be used as this family's a member, also can be used for producing fusion rotein with other albumen, such as producing fusion rotein with immunoglobulin (Ig).In addition, inventor OB-RGRP2 can also merge with other members of this family or exchange fragment, to produce new albumen.For example the N end of inventor OB-RGRP2 and the N end of people OB-RGRP are exchanged, to produce the albumen that new activity is higher or have new features.

At the antibody of inventor OB-RGRP2, be used to screen other members of this family, perhaps be used for affinity purification associated protein (as other members of this family).

For example, inventor OB-RGRP2 nucleic acid (encoding sequence or antisense sequences) can be introduced into cell, with expression level that improves people OB-RGRP2 or the overexpression that suppresses people OB-RGRP2.People OB-RGRP2 albumen of the present invention or its active polypeptide fragment can be applied to patient, with treatment or alleviate because of people OB-RGRP2 disappearance, no function or cause unusually related disorders, especially more relevant with obesity illness arranged.In addition, can also be with carrying out relevant diagnosis or prognosis judgement based on nucleotide sequence of the present invention or antibody.

Embodiment 2: with RT-PCR method clone OB-RGRP2

With the total RNA of fetus brain cell is template, with oligo-dT is that primer carries out the synthetic cDNA of reverse transcription reaction, behind the test kit purifying with Qiagen, carry out pcr amplification with following primer: forward primer F15 '-CCGCCGTAGCGCGTCTTG-3 ' is positioned at the section start of SEQ ID No.1; Reverse primer R1:5 '-GCATAGCAATTATTTTCCAG-3 ' is positioned at the 1532-1551bp of SEQ ID No.1.The condition of amplified reaction: in the reaction volume of 50 μ l, contain 50mmol/L KCl, 10mmol/L Tris-Cl, (pH8.5), 1.5mmol/LMgCl2,200 μ mol/L dNTP, 25pmol primer, the Taq archaeal dna polymerase of 2.5U.On PE9600 type DNA thermal cycler by 25 cycles of following conditioned response: 94 ℃ 30 seconds; 55 ℃, 30 seconds; 72 ℃ 2 minutes.When RT-PCR, establish the blank negative contrast of positive contrast of β-actin and template simultaneously.Behind the amplified production QLAGEN test kit purifying, be connected to (Invitrogen) on the pCR carrier with TA clone test kit, and measure dna sequence dna.The 1-1551bp of the dna sequence dna of PCR product and SEQ ID NO:1 is identical as a result.

Embodiment 3: the vivoexpression of reorganization OB-RGRP2, separate and purifying

Designed a pair of primer at the initiator codon place and the terminator codon place of OB-RGRP2 gene respectively, its 5 ' end has BamHI and EcoRI restriction enzyme site respectively.With plasmid 018e11 or commercially available cDNA library is that template is carried out pcr amplification acquisition OB-RGRP2 gene coding region.The process enzyme is cut amplified fragments is inserted expression vector pGEX-2T (available from Pharmacia Biotech), and transformed into escherichia coli E.coli DH5 α, containing on the LB flat board of penbritin and IPTG, 5 recombinant conversion of screening white carry out dna sequence analysis, and the genes encoding region sequence among result and the 018e11 is identical.This recombinant clone can be expressed the GST-OBRGRP2 fusion rotein, and the clone is designated as pOBRGRP2.

Choose ring bacillus coli DH 5 alpha (pOBRGRP2) bacterial strain, be inoculated in 20ml LB substratum and (contain penbritin 100 micrograms/ml), 37 ℃ of shaking culture are spent the night as seed liquor, get seed liquor and transfer in 4 liters of LB substratum by 2% inoculum size, and 37 ℃ of shaking culture are to thalline A ₆₀₀=0.7 o'clock (logarithmic phase), add IPTG to final concentration 0..4mmol/L, cultivate 25 ℃ again and cultivated centrifugal collection thalline 12 hours, wash with buffer A (16mMNa2HPO4 with 1 * PBS, 4mM NaH2PO4 pH6.5) presses the dissolving of 10ml/ gram thalline, ultrasonication in the ice bath, supernatant is collected in centrifugal back, supernatant liquor is crossed gsh-Sepharose 4B chromatography column, after buffer A drip washing, carries out wash-out with the elutriant that contains the 5mM reduced glutathione.Elutriant shows that through SDS-PAGE there is the GST-OBRGRP2 fusion protein product at about 44kDa place.

Embodiment 4: anti-OB-RGRP2 production of antibodies

With the synthetic specific polypeptide of following OB-RGRP2: the NH2-Leu-Pro-Ile-Val-Phe-Ala-Arg-Ala-His-Leu-COOH of Peptide synthesizer (PE-ABI).This polypeptide formed mixture with hemocyanin and bovine serum albumin coupling respectively, and method is referring to Avrameas.Immunochemistry, 1969; 6:43.Add the complete Freund's adjuvant immunizing rabbit with the above-mentioned hemocyanin polypeptide complex of 4mg, add the incomplete Freund's adjuvant booster immunization once with the hemocyanin polypeptide complex again after 15 days.Employing is done the titre that ELISA measures antibody in the rabbit anteserum through the titer plate of 15 μ g/ml bovine serum albumin polypeptide complex bag quilts.From the rabbit anteserum of antibody positive, separate total IgG with albumin A-Sepharose.Polypeptide is incorporated on the Sepharose 4B post of cyanogen bromide-activated, from total IgG, separates anti-peptide antibody with affinity chromatography.Immuno-precipitation proof antibody purified can combine with OB-RGRP2 specifically.

All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.

Sequence table (1) general information: (i) applicant: Shengyuan Gene Development Co., Ltd., Shanghai is denomination of invention (ii): new protein correlative to obesity inhibin receptor and encoding sequence, and (iii) sequence number of method for making and purposes: the information of 2 (2) SEQ ID NO.1: (i) sequence signature

(A) length: 2701bp

(B) type: nucleic acid

(C) chain: strand

( D ) ： ( ii ) ：cDNA. ( xi ) ：SEQ ID NO.1 1 CCGCCGTAGC GCGTCTTGGG TCTCCCGGCT GCCGCTGCTG CCGCCGCCGC 51 CTCGGGTCGT GGAGCCAGGA GCGACGTCAC CGCCATGGCA GGCATCAAAG101 CTTTGATTAG TTTGTCCTTT GGAGGAGCAA TCGGACTGAT GTTTTTGATG151 CTTGGATGTG CCCTTCCAAT ATACAACAAA TACTGGCCCC TCTTTGTTCT201 ATTTTTTTAC ATCCTTTCAC CTATTCCATA CTGCATAGCA AGAAGATTAG251 TGGATGATAC AGATGCTATG AGTAACGCTT GTAAGGAACT TGCCATCTTT301 CTTACAACGG GCATTGTCGT GTCAGCTTTT GGACTCCCTA TTGTATTTGC351 CAGAGCACAT CTGATTGAGT GGGGAGCTTG TGCACTTGTT CTCACAGGAA401 ACACAGTCAT CTTTGCAACT ATACTAGGCT TTTTCTTGGT CTTTGGAAGC451 AAGGACGACT TCAGCTGGCA GCAGTGGTGA AAAGAAATTA CTGAACTATT501 GTCAAATGGA CTTCCTGTCA TTTGTTGGCC ATTCACGCAC ACAGGAGATG551 GGGCAGTTAA TGCTGAATGG TATAGCAAGC CTCTTGGGGG TATTTTAGGT601 GCTCCCTTCT CACTTTTATT GTAAGCATAC TATTTTCACA GAGACTTGCT 651 GAAGGATTAA AAGGATTTTC TCTTTTGGAA AAGCTTGACT GATTTCACAC 701 TTATCTATAG TATGCTTTTT GTGGTGTCCT GCTGAATTTA AATATTTATG 751 TGTTTTTCCT GTTAGGTTGA TTTTTTTTGG AATCAATATG CAATGTTAAA 801 CACTTTTTTA ATGTAATCAT TTGCATTGGT TAGGAATTCA GAATTCCGCC 851 GGCTCTATTA CTGGTCAAGT ACATCTTTTC TCTTAAAATT ATTTAGCCTC 901 CATTATTACA AAAAATTATA AAAATAAGTT TTCAGTCAGT CAGGATGACA 951 TCACTCCCAA TGTTATGCAG ACATACAGAC GGTTGGCATA CGTTATAGAC1001 TGTATACTCA GTGCAAATAT AGCTGCATTT ATACCTCAGA GGGGCCAAGT1051 GTTAATGCCC ATGCCCTCCG TTAAGGGTTG TTGGTTTTAC TGGTAGACAG1101 ATGTTTTGTG GATTGAAAAT TATTTTATGG AATTGCTACA GAGGAGTGCT1151 TTTCTTCTCA ATTGTTAGAA GAATTTATGT TAAACTTTAA GGTAAGGGTG1201 TAAAAACATT TTTGAGATAA GGTTTTTATT TATGTTTATT ATTGTTAGAG1251 TGAGTTGCAA TGTGGGAAGA AATGACATTG AAATTCCAGT TTTTGAATCC1301 TGTTTCTATT TATAAGTGAA ATTTGTGATC TCCTATCAAC CTTTCATGTT1351 TTACCCTGTT AAAATGGACA TACATGGAAC CACTACTGAT GAGGGACAGT1401 TGTATGTTTG CATCATATAT GCCAGAAAAC CTTCCTCTGC TTCCTCCTTT1451 TGACTTATTT GGTATGTTGT ATATATTACA TAAAATAACT TTTCAAATAT1501 AGTTTAATAA CACTTAGAAG TGTTTACTTA CCTGGAAAAT AATTGCTATG1551 CCGTACATTC AGAGTGCCCC CTCCCCTGCA AGGCCTTGCC ATGATTAACA1601 AGTAACTTGT TAGTCTTACA GATAATTCAT GCATTAACAG TTTAAGATTT1651 AGACCATGGT AATAGTAGTT CTTATTCTCT AAGGTTATAT CATATGTAAT1701 TTAAAAGTAT TTTTAAGCCA AGTTTCCTGT ATACCTCTGA ACTGTTTTGA1751 TTTTGAGTTC ATCATGATAG ATCTGCTGTT TCCTTATAAA AGGCATTTGT1801 TGTGTGAGTT AATGCAAAGT AGCCAAGTCC AGCTATATAG CAGCTTCAGA1851 AACATACCTG ACCAAAAAAT TCCCAGTAAC CAGGCATGAT CAATTTATAG1901 TGGTCGTTTA CATCTAATAA TTATCAGGAC TTTTTTCAGG AGTGGGTTAT1951 AAAAACATTC AAGTTGGTCT GACAGTATTT TGTTAAGGAT ATTTGTTTGT2001 ATGTTTATTC AGTATACTTA CATAAAAATT ATTTCGCCAT CAGCCAAAAC2051 TCAGTAATCA TGACAGCTGT CTGTTGTTTT ATGAAGTTTA TTTCTCAAGA2101 AAATGGGAAT AAATTTGGGA TTTGTTCAGC TTTTTTACTA AAGATGCCTA2151 AAGCCACAGG TTTTATTGCC TAACTTAAGC CATGACTTTT AGATATGAGA2201 TGACGGGAAG CAGGACGAAA TATCGGCGTG TGGCTGGAGC CTTCCCACTG2251 GAGGCTGAAA GTGGCTTGTG GTATTATAAT GTTCAGATTT CAAGAGGAAG2301 GTGCAGGTAC ACATGAGTTA GAGAGCTGGT GAGACAGTTG GGAACTCTTT2351 GTGCTTGTGA TCTACTGGAC TTTTTTTTTG CAGGAAGTGC ATTCTCTGGT2401 CCTTCCCTAT TTTCTGTTCT GGATGTCAGT GCAGTGCACT GCTACTGTTT2451 TATCCACTTG GCCACAGACT TTTTCTAACA GCTGCGTATT ATTTCTATAT2501 ACTAATTGCA TTGGCAGCAT TGTGTCTTTG ACCTTGTATA CTAGCTTGAC2551 ATAGTGCTGT CTCTGATTTC TAGGCTAGTT ACTTGAGATA TGAATTTTCC2601 ATAGAATATG CACTGATACA ACATTACCAT TCTTCTATGG AAAGAAAACT2651 TTTGATGATG AAACAATAAA GATTTTAAAT ATCAAAAAAA AAAAAAAAAA2701 A ( 2 ) SEQ ID NO.2： ( i )

(A) length: 131 amino acid

(B) type: amino acid

( D ) ： ( ii ) ： ( xi ) ：SEQ ID NO.2 1 Met Ala Gly Ile Lys Ala Leu Ile Ser Leu Ser Phe Gly Gly Ala 16 Ile Gly Leu Met Phe Leu Met Leu Gly Cys Ala Leu Pro Ile Tyr 31 Asn Lys Tyr Trp Pro Leu Phe Val Leu Phe Phe Tyr Ile Leu Ser 46 Pro Ile Pro Tyr Cys Ile Ala Arg Arg Leu Val Asp Asp Thr Asp 61 Ala Met Ser Asn Ala Cys Lys Glu Leu Ala Ile Phe Leu Thr Thr 76 Gly Ile Val Val Ser Ala Phe Gly Leu Pro Ile Val Phe Ala Arg 91 Ala His Leu Ile Glu Trp Gly Ala Cys Ala Leu Val Leu Thr Gly106 Asn Thr Val Ile Phe Ala Thr Ile Leu Gly Phe Phe Leu Val Phe121 Gly Ser Lys Asp Asp Phe Ser Trp Gln Gln Trp

Claims (10)

1. isolated nucleic acid molecule is characterized in that it comprises: coding has the nucleotide sequence of the polypeptide of people OB-RGRP2 protein-active, and the nucleotide sequence of 85-480 position has 70% homology at least among this nucleotide sequence and the SEQ ID No.1; Perhaps this nucleotide sequence can be under the height stringent condition with SEQ ID No.1 in the nucleotide sequence hybridization of 85-480 position.

2. molecule as claimed in claim 1 is characterized in that, this nucleotide sequence coded polypeptide with aminoacid sequence shown in the SEQ ID No.2.

3. molecule as claimed in claim 1 is characterized in that, this nucleotide sequence has the sequence of 85-480 position among the SEQ ID No.1.

4. isolating OB-RGRP2 polypeptide, it comprises: polypeptide or its conservative property variation polypeptide or its active fragments or its reactive derivative with SEQ ID No.2 aminoacid sequence.

5. polypeptide as claimed in claim 4 is characterized in that, this polypeptide is the polypeptide with SEQ ID No.2 aminoacid sequence.

6. a carrier is characterized in that, it contains the described nucleic acid molecule of claim 1.

7. one kind with the described carrier transformed host cells of claim 6.

8. preparation method with polypeptide of people OB-RGRP2 protein-active is characterized in that this method comprises:

(a) nucleotide sequence that coding is had a polypeptide of OB-RGRP2 protein-active operationally is connected in expression regulation sequence, form the OB-RGRP2 protein expression vector, the sequence from Nucleotide 85-480 position among described nucleotide sequence and the SEQ ID NO.1 has at least 70% homology;

(b) change the expression vector in the step (a) over to host cell, form the proteic reconstitution cell of OB-RGRP2;

(c) be fit to express under the condition of OB-RGRP2 protein polypeptide the reconstitution cell in the culturing step (b);

(d) isolate polypeptide with OB-RGRP2 protein-active.

9. energy and the described OB-RGRP2 polypeptid specificity of claim 4 bonded antibody.

10. nucleic acid molecule, it contains successive 10-2000 Nucleotide in the described nucleic acid molecule of claim 1.

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