CN1099423C - Human neuropeptide receptor - Google Patents

Abstract

The present invention discloses human neuropeptide receptor polypeptide, a DNA (RNA) for encoding the polypeptide, and a method for generating the polypeptide by reorganization technology. The present invention also discloses a method by which the agonists and the antagonists of the polypeptide are identified by the polypeptide, and a method by which the agonists and the antagonists are respectively and therapeutically used for treating diseases which are corresponding to the defective expression and the excessive expression for selectively describing neuropeptide receptor polypeptide. The present invention also discloses a horizontal diagnostic method for detecting the jump in a neuropeptide receptor nucleotide sequence and the change of a soluble form of the receptor.

Description

Human neuropeptide receptor

The purposes of the polynucleotide that the present invention relates to differentiate recently, polypeptide, these polynucleotide and polypeptide and the production method of these polynucleotide and polypeptide by these polynucleotide encodings.Polypeptide of the present invention is that people 7-changes film G-protein linked receptor.More particularly, polypeptide of the present invention is neuropeptide receptor polypeptide (after this being called the neuropeptide receptor polypeptide sometimes).The present invention also relates to suppress the method for the effect of these polypeptide.

Obesity is a prevailing nutritive disease in Western society.Three individual weights among ten adult Americans surpass at least 20% (Burroa, the M. New York Times, 19947 months 17 days) of their ideal body weight.The body weight that increases is an important public health problem, because it and type ii diabetes, hypertension, hyperlipoidemia and some related to cancer (Grundy, S.M. and Barnett, J.P., Disease-a-Month, 36:645-696 (1990)).

Existing (Priedman, the J.M.﹠amp of describing of 5 term single gene sudden changes in the mouse ob gene (ob) of the phenotype that causes fat; Leibel, R.L., cell, 66:217-220 (1990)).The clone of mouse ob gene and its human homologue be in the news with order-checking (Zhang, Y., etc., nature, 372:425-431 (1994)).The ob genes encoding has the 4.5-kb fatty tissue mRNA of 167 amino acid whose open reading frame of high conservative.The aminoacid sequence of prediction is 84% identical between the mankind and mouse, and has the feature of secretory protein.The part that the ob gene product can be used as the fatty tissue signal pathway works, its play a role in order to regulate body fat to accumulate (the same, 425).

In the brain district that involves feed behavior adjusting, hypothalamic ventromedial nucleus (VMH) is considered to most important full center in the central nervous system (CNS).Therefore the energy balance of supposition in Mammals is fed-encircles control, the amount of the energy of being stored is by the hypothalamus perception therein, its adjusting food intake and energy expenditure are to keep constant body temperature (Ombeck, J.R., the biomedical magazine of Yale, 20:545-552 (1948) and Kennedy, G.C., Proc.R.Soc.148:578-592 (1953)).Smoulder at fat in (lipostasis) theory, how much what body fat accumulated is regulated by CNS, and the metabolic product of body fat influences energy balance (Kennedy, G.C., Proc.R.Soc.148:578-592 (1953)) by interacting with hypothalamus.

Can not from fat differentiate the signal obstacle infer fat smoulder theoretical confirmation.The component of at least a signalling system round-robin possibility in blood flow proposes (Dietrich by Hervey the earliest, W. etc., genetics, 131:423-447 (1992)), he has shown that by blood vessel graft blood being transferred to (parabiosis experiment) the untreated animal from the animal with VMH damage causes weakening of in the animal that does not have wound ingestion of food.Be apparent that to have the ob gene product now, show ob this repetition factor of can encoding by the excretory evidence.

The ob signal can act on the CNS directly or indirectly, suppresses ingestion of food and/or regulates energy expenditure with the part as homeostatic mechanism, keeps the homoeostasis (Zhang of fat mass, V etc., nature, 372:425-431,431 (1994)).The ob gene fatty excretory protein of obviously encoding, and sudden change stops the translation or the expression (Rink, T., nature, 372:406-407 (1994)) of described gene significantly.

The parabiosis experiment shows that the ob acceptor is by mouse db (diabetes) genes encoding (Coleman, D.L., Diabetologia, 14:141-149 (1978)).Mouse with the sudden change in the db gene is also fat, and having possibility is acceptor damaged damaged (the same, 406).

Neuropeptide tyrosine is similar to ob gene product part and is its adjusting feed reaction.Neuropeptide tyrosine acts on and is called as Y ₁, Y ₂, Y ₃With atypical Y ₁On at least four types the Neuropeptide Y Receptors of acceptor (it regulates the feed reaction that is stimulated by neuropeptide tyrosine).

Neuropeptide tyrosine has biological function miscellaneous.Find that neuropeptide tyrosine is distributed widely in central nervous system (CNS) and the peripheral nervous system (PNS), in PNS, find in the neurone of neuropeptide tyrosine in the neural distribution of blood vessel norepinephrine energy sympathetic nerve and other smooth muscle tissue and in the enteric nervous system.Neuropeptide tyrosine immunoreactivity fiber also appears in the non-vascular smooth muscle, round exocrine gland and superficial epithelium.Neuropeptide tyrosine also appears in the neurone subgroup, general and other neurohumor (specific norepinephrine) co.

In CNS, neuropeptide tyrosine is included in the GABA energy relay cell of higher center and advantage catecholamine energy cell (it protrudes backly).For example, neuropeptide tyrosine is included in the relay cell in skin, hippocampus, tonsilla, base portion forebrain and the striatum, and in people's brain stem, neuropeptide tyrosine is included among medullary substance and locus coeruleus (locus coeruleus) A1 and the noradrenergic neuron of A2 group in (LC).In hypothalamus, neuropeptide tyrosine is found at arc nuclear and lateral hypothalamus significantly.

In peripheral nervous system, neuropeptide tyrosine is present in the sympathetic nerve of neuroganglion rear portion, and as mentioned above, with other neurohumor (comprising catecholamine) co.When using on pharmacology, neuropeptide tyrosine demonstrates effective vasoconstrictor activity, and strengthens the vasoconstriction by many other pressure agent generations dramatically.Particularly, find that neuropeptide tyrosine at the sympathetic nerve middle and high concentration makes crown, brain and kidney portion vasoconstriction, and when being integrated into these vescular beds, neuropeptide tyrosine causes the vasoconstriction that is not reversed by adrenergic blocking drug that prolongs.These observationss cause producing a kind of like this viewpoint: neuropeptide tyrosine is candidate's medium of pathology vasospasm (when relating to crown and during cerebral vessels, being main morbidity and dead factor).

As if neuropeptide tyrosine also involve the interaction with renin-angiotensin system.In renal cortex nearby bleeding pipe bead organ, find to contain the neuropeptide tyrosine of sympathetic nerve terminal, and neuropeptide influences feritin release.The stress reaction of the real example of all time length of neuropeptide tyrosine concentration and the described peptide of infusion causes deriving from a kind of like this conclusion in these information and the hypertension animal model: this peptide involves hypertension.

As if in central nervous system, neuropeptide tyrosine mainly is positioned within the relay cell, have regulating effect this its.Therefore, it has widely and different effects, comprises memory impairments and the possible effect in Alzheimer's.Neuropeptide tyrosine is to cause the most effective known substance that increases feed, can work in the hereditary basis of type ii diabetes.Neuropeptide tyrosine also can be used as conditioning agent in compressing reaction and the reproductive function and pituitary gland function and potential dysfunction of nervous regulation and plays a role.

According to one aspect of the present invention, the invention provides new mature polypeptide with and bioactive and and the diagnosis on or the treatment on useful fragment, analogue and derivative.Polypeptide of the present invention is a human origin.

According to another aspect of the present invention, the invention provides the isolated nucleic acid molecule of coding receptor polypeptides of the present invention, comprise mRNA, DNA, cDNA, genomic dna with and antisense analogue and its biologic activity and at useful fragment and derivative in the diagnosis or in the treatment.

According to another aspect of the present invention, the invention provides the method that produces this receptor polypeptides through recombinant technology, this method is included under the condition that promotes receptor polypeptides expression of the present invention, cultivation contains the reorganization protokaryon and/or the eukaryotic host cell of the nucleotide sequence of the described receptor polypeptides of encoding, and then reclaims said polypeptide.

According to another aspect of the present invention, the invention provides the antibody of these polypeptide.

According to another aspect of the present invention, the invention provides screening and be attached on the receptor polypeptides of the present invention and activate this polypeptide or suppress the method for the activated compound of this polypeptide.

According to another embodiment of the present invention, the invention provides for stimulating neuronal growth, regulate neurotransmission, the availability of the food of enhanced activity level and picked-up, method to host's administered compound, described compound is attached on the receptor polypeptides of the present invention and activates this polypeptide, and this polypeptide is useful in preventing and/or treating obesity, hyperlipemia and some cancer.

According to another aspect of the present invention, the invention provides the method for using described receptor polypeptides through gene therapy, with the relevant illness of expression deficiency of treatment with the part of the overexpression of receptor polypeptides of the present invention or described receptor polypeptides.

According to another embodiment of the present invention, the invention provides method to host's administered compound, described compound is attached on the receptor polypeptides of the present invention and suppresses the activation of this polypeptide, it is preventing and/or treating Alzheimer's, type ii diabetes, epilepsy, anxiety, anxiety, hypertension, is useful in cardiovascular diseases, psychosis and the obesity that caused by neuropeptide tyrosine.

According to another aspect of the present invention, the invention provides nucleic acid probe, this nucleic acid probe comprises length and is enough to specifically nucleic acid molecule with coding polynucleotide sequence hybridization of the present invention.

According to another aspect of the present invention, the invention provides the diagnostic measurement method of level of change of the soluble form of relevant disease of sudden change in the nucleotide sequence that detects with coding polypeptide of the present invention and detection receptor polypeptides.

According to another aspect of the present invention, the invention provides these receptor polypeptides, or the method for the relevant purpose of the external synthetic that is used for and dna vector synthetic with scientific research, DNA of the polynucleotide of these polypeptide of encoding.

From the instruction of this paper, those skilled in the art can know these and other aspect of the present invention.

Following accompanying drawing is used for illustrating embodiment of the present invention, and has no intention to be used for limiting the included scope of claim of the present invention.

Fig. 1 has shown the cDNA sequence and the corresponding deduced amino acid of neuropeptide receptor polypeptide of the present invention.Used the abbreviation of amino acid standard single-letter.Adopt 373 automated DNA sequenators (Applied Biosystem company) to check order.

Fig. 2 has shown the cDNA sequence and the corresponding deduced amino acid of neuropeptide receptor splice variant 1 polypeptide of the present invention.Used the abbreviation of amino acid standard single-letter.

Fig. 3 has shown the cDNA sequence and the corresponding deduced amino acid of neuropeptide receptor splice variant 2 polypeptide of the present invention.Used the abbreviation of amino acid standard single-letter.

Fig. 4 has illustrated aminoacid sequence and 7 commentaries on classics film districts of described neuropeptide receptor.There is underscore in described commentaries on classics film district, and represents with TM.

Fig. 5 has illustrated aminoacid sequence and 7 commentaries on classics film districts of described neuropeptide receptor splice variant 1.There is underscore in described commentaries on classics film district, and represents with TM.

Fig. 6 has illustrated aminoacid sequence and 7 commentaries on classics film districts of described neuropeptide receptor splice variant 2.There is underscore in described commentaries on classics film district, and represents with TM.

Fig. 7 has shown human neuropeptide receptor polypeptide (and human neuropeptide Y of the present invention ₁Acceptor) amino acid identity between.

Receptor polypeptides of the present invention is some acceptors known and unknown part, and said part is regulated The unify work of the peripheral tissues that regulated by the central nervous system cell among both of central nervous system The property. The example of this part is neuropeptide tyrosine, Substance P, people ob gene outcome and neural sharp Peptide B. Therefore, the adjusting of receptor polypeptides activity of the present invention can have and treats widely and diagnose Purposes is particularly for the treatment of diabetes.

The inventor has separated the full length cDNA clone of the human neuropeptide receptor polypeptide of encoding. The institute State full-length cDNA and be positioned on No. 1 chromosome position p31-34 of people, it is corresponding to discovery Position on No. 4 chromosomes of the mouse of db gene. It is believed that mouse db gene code obesity The acceptor of gene outcome.

According to one aspect of the present invention, the invention provides a kind of nucleic acid (multinuclear glycosides of separation Acid), this nucleic acid coding has the amino acid sequence (SEQ ID NO:2) of the derivation of Fig. 2 Mature polypeptide or by April 27 nineteen ninety-five with the clone's of preserving number ATCC_____ preservation The mature polypeptide of cDNA coding.

Polynucleotides of the present invention find in the hypothalamic cDNA library deriving from the adult, its Structurally relevant with g protein coupled receptor family, described neuropeptide receptor polypeptide comprises one The ORF of the protein of 402 amino acid residues of coding. Described neuropeptide receptor protein Demonstrate and human neuropeptide receptor Y₁The homology of receptor protein top is at whole amino Have 52% similitude and 26% homogeny on the acid sequence.

Polynucleotides of the present invention can be rna form or dna form, and wherein DNA comprises CDNA, genomic DNA and synthetic DNA. This DNA can be two strands or strand, and If strand then can be coding strand or non-coding (antisense) chain. This encoding mature polypeptide Coded sequence can with coded sequence (SEQ ID NO:1) shown in Figure 1 or the clone of preservation Coded sequence identical; Because Feng Yu or the degeneracy of genetic code, this coded sequence also Can be a kind of different coded sequence, DNA (SEQ ID NO:1) or the institute of its energy and Fig. 1 Say the identical mature polypeptide of cDNA coding of preservation thing.

The mature polypeptide of code pattern 2 (SEQ ID NO:2) or the described said preservation thing of coding The polynucleotides of the mature polypeptide of cDNA coding can comprise: only be the encoding mature polypeptide Coded sequence; The coded sequence of mature polypeptide (with dispensable additional code sequence) and non-volume The code sequence is such as 5 of introne or mature polypeptide encoded sequence ' and/or 3 ' non-coding sequence.

Like this, " polynucleotides of coded polypeptide " this term comprises and only contains the peptide coding order Row polynucleotides and also contain additional coding and/or the polynucleotides of non-coding sequence.

The invention still further relates to above-described polynucleotides variant, this variant coding has Fig. 2 Derivation amino acid sequence (SEQ ID NO:2) polypeptide or by the clone's of preservation cDNA Fragment, analog and the derivative of the polypeptide of coding. This polynucleotides variant can be natural The polynucleotides variant that the polynucleotides allelic variant that produces or non-natural produce.

Like this, the present invention includes coding identical mature polypeptide (SEQ ID NO:2) as shown in Figure 1 Or by the polynucleotides of the identical mature polypeptide of the clone's of preservation cDNA coding, and coding Mature polypeptide shown in Fig. 1 (SEQ ID NO:2) or by the clone's of preservation cDNA coding The polynucleotides variant of fragment, derivative and analog of mature polypeptide. These nucleotides become Body comprises disappearance variant, replacement variant, interpolation or inserts variant. The specific examples of this variant Comprise the polynucleotide sequence shown in SEQ ID NO:3 and 5, it encodes respectively of the present invention The splice variant 1 and 2 of polypeptide.

As above indicated, described polynucleotides can have a kind of coded sequence, this order Row are coded sequences of the clone of the coded sequence shown in Fig. 1 (SEQ ID NO:1) or preservation The allelic variant of natural generation. Allelic variant known in the art is another of polynucleotide sequence The form of kind, it can have replacement, disappearance or the interpolation of one or more nucleotides, and essence On do not change the function of coded polypeptide.

Also can the encode soluble form of neuropeptide receptor polypeptide of described polynucleotides, this polypeptide is The outer part of the born of the same parents of the polypeptide in full-length polypeptide cracking TM of the present invention and the born of the same parents behind the functional domain.

Polynucleotides of the present invention also can have the coding that merges with flag sequence in frame Sequence, described flag sequence is so that can purifying polynucleotides of the present invention. Bacterial host Situation under, described flag sequence can be six histidine marks that provided by the pQE-9 carrier, It is used for the mature polypeptide that purifying and mark merge, perhaps for example when using mammalian cell When (such as the COS-7 cell), flag sequence can be hemagglutinin (HA) mark. Said The HA mark is corresponding to a kind of epi-position that derives from influenza hemagglutinin protein (Wilson, I., etc., cell, 37:767 (1984)).

(condition is two to the invention still further relates to polynucleotides with sequence hybridization described above Have at least 70% between the sequence, preferably have at least 90%, more preferably have at least 9 5% Identity property). The present invention be more particularly directed under stringent condition assorted with above-described polynucleotides The polynucleotides of handing over. As used herein, term " stringent condition " refers to only have between sequence At least 95%, hybridization just can take place when preferably having at least 97% homogeny. At one In the preferred embodiment, with the polynucleotide encoding of above-described multi-nucleotide hybrid like this One peptide species, it keeps in fact and cDNA (SEQ ID NO:1) or said guarantor by Fig. 1 Hide identical biological function or the activity of mature polypeptide of cDNA (s) coding of thing, that is, by Keep bind receptor part ability (even described polypeptide can not be as film in conjunction with neuropeptide Acceptor works), for example by causing second messenger's reaction, as a kind of nerve of solubility The peptide acceptor works.

In addition, described polynucleotide can tool some polynucleotide like this, it has at least 20 bases, preferably at least 30 bases more preferably are at least 50 bases, itself and multi-nucleotide hybrid of the present invention, and has the homogeny with these polynucleotide as mentioned above, not retentive activity.This polynucleotide can for example or as diagnostic probe or as the PCR primer be used to reclaim polynucleotide as the probe of SEQ ID NO:1 polynucleotide or its variant.

The mentioned preservation thing of this paper will keep according to the regulation of the microbial preservation budapest treaty of the international recognition that is used for patented procedure.These keep thing only is in order to provide convenience to those skilled in the art, is not 112 required preservations of 35U.S.C.Be included in the described preserved material polynucleotide sequence and by its amino acid sequence coded this paper reference in the lump, and be used to solve any contradiction on this paper sequence description.Any manufacturing, use or sale to preserved material need not give any such permission through permission at this.

The invention still further relates to polypeptide, its have Fig. 2 deduced amino acid (SEQ ID NO:2) the FGF polypeptide or have polypeptide, and the fragment of this peptide species, analogue and derivative by the cDNA amino acid sequence coded of said preservation thing.

Term " fragment ", " derivative " and " analogue " during when the polypeptide (SEQ ID NO:2) of relevant Fig. 2 or by the cDNA encoded polypeptides of said preservation thing, refer to the biological function or the active polypeptide that keep identical with such polypeptide basically.That is, the part by keeping bind receptor ability (even described polypeptide can not work in conjunction with neuropeptide receptor as film).Like this, analogue comprises proteinogen, and this analogue can partly excise and then produce active mature polypeptide by proteinogen and activate.Specific example is respectively the splice variant 1 and 2 of Fig. 2 and 3 (SEQ ID NO:4 and 6).

Polypeptide of the present invention can be a recombinant polypeptide, natural polypeptides or synthetic polypeptide, preferably recombinant polypeptide.

The polypeptide of said Fig. 2 (SEQ ID NO:2) or by the fragment of the cDNA encoded polypeptides of said preservation thing, derivative or analogue can be: (i) a kind of like this, wherein one or more amino-acid residues can be also can not be by genetic codon amino acids coding residue by the amino-acid residue that conservative or non-conservative amino acid residues replaces (preferably conservative amino acid residues replacement) replacement and replacement, perhaps (ii) a kind of like this, wherein one or more amino-acid residues comprise substituting group, perhaps (iii) a kind of like this, wherein mature polypeptide and another kind of compound merge, described compound is as increasing the polypeptide compound (for example polyoxyethylene glycol) of half life, perhaps (iv) a kind of like this, wherein additional amino acid and mature polypeptide merge, for example a kind of like this sequence, it is to be used for the sequence of purifying mature polypeptide, the perhaps (iv) splice variant of mature polypeptide, this variant can have the one or more aminoacid deletion from mature polypeptide.By the elaboration of this paper, can think that such fragment, derivative and analogue is within those skilled in the art's ken.

The present invention preferably provides the polypeptide and the polynucleotide of unpack format, and preferably described polypeptide is become homogeneous with the polynucleotide purifying.

Term " gene " is meant the DNA section relevant with producing polypeptide chain; It comprises zone and zone afterwards (leader and tailer sequence) and the intervening sequence (intron) between each coding section (exon) before the coding region.

Term " isolating " means described material and has broken away from its primal environment (for example, natural surroundings is if it is natural generation).For example, a kind of polynucleotide or polypeptide that is present in the natural generation in the animal alive is not isolating, but with natural system in the material of some or all coexistence identical polynucleotide or the polypeptide that separate be isolating.Such polynucleotide can be the parts of carrier, and/or such polynucleotide or polypeptide can be the part of composition, and it remains isolating, and this is because this carrier or composition are not the parts of its natural surroundings.

The present invention also relates to comprise the carrier of polynucleotide of the present invention and the host cell that produces through genetically engineered with carrier of the present invention, and the method that produces polypeptide of the present invention through recombinant technology.

Through genetically engineered operation (transduction, conversion or transfection), said carrier can be cloning vector or expression vector to host cell with carrier of the present invention.This carrier for example can be, forms such as plasmid, virion and phage.The through engineering approaches host cell can modified to such an extent that be suitable for activating promotor, select to cultivate in the conventional nutritional medium of transformant or amplification human neuropeptide receptor gene.Culture condition, for example temperature and pH value etc. are those of host cell that were used to express selection in the past, are conspicuous to those of ordinary skill.

Polynucleotide of the present invention can be used for producing polypeptide through recombinant technology.Like this, for example, polynucleotide can be included in any of the various expression vectors that are used for express polypeptide.Such carrier comprises karyomit(e), non-chromosome and synthetic DNA sequence, for example SV 40 derivatives; Bacterial plasmid; Phage DNA; Baculovirus; Yeast plasmid; Make up deutero-carrier, viral DNA (as cowpox, adenovirus, fowl avipoxvirus and pseudorabies virus) from plasmid and phage DNA.Yet any other carrier also can use, as long as it is reproducible and stable in the host.

Can suitable dna sequence dna be inserted in the carrier with several different methods.In general, with methods known in the art dna sequence dna is inserted into suitable restriction endonuclease site.Such method and other method are considered in those skilled in the art's ken.

Said dna sequence dna is operably connected to suitable instructing on the mRNA synthetic expression control sequenc (promotor) in expression vector.The representative example of such promotor can should be mentioned that: LTR or SV 40 promotors, colibacillary lac or trp, phage P _LPromotor and known other promotor that controlling gene is expressed in protokaryon or eukaryotic cell or their virus.Said expression vector also comprises the ribosome bind site that is used for translation initiation and Transcription Termination.This carrier also can comprise the suitable sequence of expressing for amplification.

In addition, expression vector preferably comprises one or more selected markers, it is provided for selecting the phenotypic characteristic (for example Tetrahydrofolate dehydrogenase of eukaryotic cell culture or neomycin resistance, or for example tsiklomitsin and the amicillin resistance in the intestinal bacteria) of transformed host cells.

Comprise above-described suitable dna sequence dna and suitable promotor or the carrier of control sequence and can be used to transform suitable host, so that it can marking protein.

As the representative example of suitable host, can should be mentioned that here: bacterial cell, as intestinal bacteria, streptomycete, Salmonella typhimurium; The fungal cell is as yeast; Insect cell such as Drosorphila S2 and Spodoptera Sf9; Zooblast such as CHO, COS or Bowes melanoma; Adenovirus; Vegetable cell etc.By the elaboration of this paper, within the ken that is chosen in those skilled in the art to suitable host.

More particularly, the present invention also comprises recombinant precursor, and this construct comprises above broadly described one or more sequences.This construct comprises carrier, and as the carrier of plasmid or virus, this carrier has inserted sequence of the present invention forward or backwards.Under the even more ideal situation of this embodiment, construct also comprises the adjusting sequence that can be operationally connected on the described sequence, comprises for example promotor.A large amount of carrier and promotors that are fit to are well known by persons skilled in the art, and are obtainable by commercial sources.Provide following carrier by way of example: bacterium: pQE70, pQE60, pQE-9 (Qiagen), pbs, pD10, phagescript, psiX174, pbluescript SK, pbsks, pNH8A, pNH16a, pNH18A, pNH46A (Stratagene), ptrc99a, pKK223-3, pKK233-3, pDR540, pRITS (Pharmaca); Eucaryon: pWLNEO, pSV2CAT, pOG44, pXT1, pSG (Stratagene), pSVK3, pBPV, pMSG, pSVL (Parmacia).Yet any other plasmid or carrier can use, as long as they are reproducible and stable in the host.

Can be with CAT (CAT) carrier or other carrier that has selected marker from any required gene Selection promoter region.Two kinds of suitable carriers are PKK232-8 and PCM7.The bacterium promotor of mentioning especially comprises lacI, lacZ, T3, T7, gpt, λ P _R, P _LAnd trp.Promoter in eukaryote comprises that CMV is early stage immediately, HSV thymidine kinase, early stage and late period SV40, derive from retroviral LTRs and mouse metallothionein(MT)-I.Within the level that is chosen in those of ordinary skills to appropriate carriers and promotor.

In another embodiment, the present invention relates to comprise the host cell of the above construct.Said host cell can be higher eucaryotic cells (as a mammalian cell), or eukaryotic cell (as yeast cell) such as low, and perhaps host cell can be prokaryotic cell prokaryocyte (as a bacterial cell).Can be by the transfection of calcium phosphate transfection, DEAE-dextran mediation, or electroporation is incorporated into construct (Davis, L., Dibner, M., Battey, I., the basic skills in the molecular biology, (1986)) in the host cell effectively.

Construct in the host cell can be used for producing in a usual manner the gene product by the recombination sequence coding.In addition, polypeptide of the present invention can produce by conventional peptide synthesizer is synthetic.

Polypeptide fragment of the present invention can be used for through the corresponding full-length polypeptide of the synthetic generation of peptide.Therefore described fragment can be as the intermediate that produces full-length polypeptide.Polypeptide fragment of the present invention can be used for synthetic full-length polypeptide of the present invention in a similar fashion.

Mature protein can be expressed under suitable promotor control in mammalian cell, yeast, bacterium or other cell.Employing derives from the RNA of DNA construct of the present invention, and cell free translation system also can be used for producing this protein.Sambrook etc., molecular cloning: laboratory manual, second edition, the cold spring port, N.Y., the suitable clone and the expression vector that use with protokaryon and eucaryon host have been described in (1989) (this paper is reference in the lump).

The encode enhancer sequence that is inserted in the carrier of transcribing of DNA of polypeptide of the present invention of higher eucaryote strengthens.Enhanser is the cis-acting elements of DNA, and usually about 10 to 300bp, and acting on increases it and transcribe on the promotor.Example comprises polyoma enhanser and the adenovirus enhanser on SV 40 enhansers on the replication orgin side 100 to 270bp in late period one, cytomegalovirus early promoter enhanser, the replication orgin side in late period one.

In general, recombinant expression vector comprises replication orgin and allows the selected marker (for example, intestinal bacteria ampicillin resistance gene and Saccharomyces cerevisiae TRP1 gene) of host cell conversion and the promotor that instructs the downstream configurations sequence to transcribe that comes from cance high-expression gene.Such promotor can be come the operon of own coding glycolytic ferment (for example glycerol 3-phosphate acid kinase (PGK)), α-factor, acid phosphatase or heat shock protein etc.The allos structure sequence is with suitable manner (phase) and translation initiation and terminator sequence assembling, and preferably, the leader sequence that advances periplasmic space or extracellular substratum with the protein secreting that can instruct translation assembles.Heterologous sequence can be also encoding fusion protein not, this protein comprises the terminal peptide of differentiating of the N-that gives required feature, required feature for example, the recombinant products of expression is stable or simplify purification step.

Structural DNA sequence by the desired protein of will encoding and suitable translation initiation and termination signal are inserted and are made up the useful expression vector that is used for bacterium with having functional promotor can operate reading method (reading phase).Said carrier comprises one or more Phenotypic Selection marks and replication orgin, to guarantee to keep carrier the host and amplification is provided when needing.The prokaryotic hosts that be fit to transform comprises various (though other also can select use) of intestinal bacteria, subtilis, Salmonella typhimurium, Rhodopseudomonas, streptomyces and Staphylococcus.

As one representational but be not restrictive example, the useful expression vector that is used for bacterium can comprise selected marker and the bacterium replication orgin that comes from commercially available plasmid (genetic elements that comprises well-known cloning vector pBR322 (ATCC37017)).Commercially available carrier like this comprises, for example, and pKK223-3 (Pharmacia Fine chemical company, Uppsala, Sweden) and GEM1 (Promega Biotec, Madison, WI, the U.S.).These pBR322 " skeleton " fragment and suitable promotor and structure sequence combination to be expressed.

Transform and appropriate host strain growth extremely after the suitable cell density at the appropriate host bacterial strain, induce the promotor of selection with suitable method (for example temperature inversion or chemical induction), other for some time of cell cultures.

Typically,, keep formed crude product to be used for further purifying through physics or chemical process smudge cells through centrifugal cell harvesting.

The microorganism cells that can be used for marking protein through the method fragmentation of any routine, described method comprise freeze-thaw cycle, sonication, Mechanical Crushing or use the lysis agent.These methods are well known to a person skilled in the art.

Various mammalian cell culture systems also can be used for express recombinant protein matter.The example of mammalian expression system comprises by the monkey kidney inoblast COS-7 clone of Gluzman (cell, 23:175 (1981)) description and other can express the clone of compatible carrier, for example C127,3T3, CHO, HeLa and bhk cell system.Mammalian expression vector comprises replication orgin, suitable promotor and enhanser, also can comprise any essential ribosome bind site, polyadenylation site, donor splicing site and acceptor site, transcription termination sequence and 5 ' flank non-transcribed sequence.The dna sequence dna that derives from SV40 montage and polyadenylation site can be used to provide required non-transcribed genetic elements.

Neuropeptide receptor polypeptide of the present invention can reclaim and purifying from the reconstitution cell culture with former employed method, and described method comprises ammonium sulfate or ethanol sedimentation, sour extraction, negatively charged ion or cation-exchange chromatography, phosphorus Mierocrystalline cellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography and phytohemagglutinin chromatography.In finishing the configuration of mature protein, can use the protein refolding step when needing.At last, can use high performance liquid chromatography (HPLC) as last purification step.

Neuropeptide receptor polypeptide of the present invention can be the product of natural purifying, or the product of chemical synthesis process, or produce from protokaryon or eucaryon host (for example insect of bacterium, yeast, higher plant, cultivation and mammalian cell) through recombinant technology.According to the host that reorganization is used in the production method, polypeptide of the present invention can be glycosylated maybe can be nonglycosylated.Polypeptide of the present invention also can comprise the initial methionine amino-acid residue.

Polynucleotide of the present invention and polypeptide can be as treatment and the Studies on Diagnosis reagent and the materials of human diseases.

Human nerve's peptide receptor polypeptides of the present invention can be used for screening in conjunction with and activate described receptor polypeptides compound method and be used for screening the method that is attached on the receptor polypeptides of the present invention and suppresses its activated compound.

In general, with isolating, the neuropeptide receptor of immobilized or cell combining form and multiple compound contact and select those and described receptors bind and compound interactional with it.By adopting radiolabeled compound of interest or can directly measuring described combination or interaction by second messenger's effect that interaction and combination from candidate compound produce.In addition, can be to the candidate compound screening assay that is at war with, wherein known ligand and compound to be tested are introduced together, and measure the compound inhibition or strengthen tagged ligand bonded ability, preferred part is with detectable reagent mark analytically, most preferably radioactivity.With regard to compound the affinity and the selectivity of the increase of receptor polypeptides of the present invention are come SCREENED COMPOUND.

A kind of such method relates to the use melanophore, and this cell is transfected to express neuropeptide receptor of the present invention.In the PCT WO 92/01810 that published on February 6th, 1992, this triage techniques has been described.

For example, suppress receptor polypeptides activated compound of the present invention, the part of described compound and known bind receptor is contacted with melanophore in order to screen.The restraining effect of the signal that part is produced shows that described compound suppresses the activation of described acceptor.

Whether produce signal (being activated receptor) by described cell and compound to be screened are contacted and detect this compound, this screening method can be used to measure in conjunction with and activate the compound of receptor polypeptides of the present invention.

Other example is included in the system that the outer pH of born of the same parents that mensuration causes by receptor activation changes and uses the cell (for example, the Chinese hamster ovary celI of transfection) of expressing receptor polypeptides of the present invention, science for example, described in 246 volumes, 181-296 (in October, 1989).For example, can and express the cells contacting of receptor polypeptides of the present invention, and can detect second messenger's reaction (for example signal transduction or pH change) compound, whether effective to determine described potential compound as activator or inhibitor.

The RNA that another example relates to coding neuropeptide receptor of the present invention is incorporated in the Xenopus ovocyte with the described acceptor of transient expression.Then, ovocyte and receptors ligand can be contacted with compound to be screened, then detect the inhibition or the increase of cellular calcium.

Another example relates at cell surface expression neuropeptide receptor polypeptide of the present invention, and wherein, described receptor polypeptides is connected on phosphatide C or the D.Representative example as these cells can should be mentioned that endotheliocyte, smooth muscle cell, embryonic kidney cells etc.Can finish described screening from the inhibition of phosphatide second signal detection receptor activation or receptor activation as mentioned above.

Another kind method relates to be measured tagged ligand and is attached to restraining effect on the cell (having neuropeptide receptor in its surface).A kind of like this method comprises with the DNA transfecting eukaryotic cells of coding neuropeptide receptor polypeptide of the present invention so that described cell at the described acceptor of its surface expression, and makes cell contact with compound in the presence of the known ligand of mark pattern.Described part can be for example use radiolabeled.Be connected to the amount of the tagged ligand on the acceptor by the radioassay of for example measuring acceptor.Be attached on the acceptor if record compound in the minimizing through being attached to the tagged ligand on the acceptor, then tagged ligand is suppressed with combining of acceptor.

Another kind of triage techniques relates at cell surface expression neuropeptide receptor polypeptide of the present invention, and wherein, described receptor polypeptides is connected to the second messenger and goes up to increase cytosolic calcium levels in the transfection CHO cell.The example of this method comprises the nucleotide sequence transfection CHO cell with coding acceptor of the present invention, so that described acceptor is at its surface expression.Then, the cell of transfection is cultivated in the reaction mixture that has mark calcium in the presence of the compound to be screened.Can measure compound by the amount of utilizing mark (for example radioactivity) to detect the mark calcium in the transporte to cells then and increase the ability that the calcium absorption was taken in or suppressed to calcium.

The compound that is attached on the specific subprovince in the CNS also can be by above method discriminating, and described subprovince is to being important by the indirect interactional specific behavior with neuropeptide receptor polypeptide of the present invention.

In order to measure ring-type AMP level in the born of the same parents, with 100 micromole's isobutyl-xanthine (ISMX; Sigma) measures ring-type AMP in 15 minutes the full cell of processing.With transfectional cell (1 * 10 ⁶/ 0.5 milliliter) with the known or unknown receptors ligand incubation of 10 micromole's forskolins and various concentration.Through adding hydrochloric acid to 0.1M, at room temperature incubation is 15 minutes, and neutralization is also carried out the diluted sample termination reaction with 50mM sodium acetate (pH6.2).Adopt the quantitative ring-type APM of radioimmunoassay.

In order to measure the cellular calcium level, transfectional cell is suspended in the sample substratum (RPMI 1640 substratum of modification/10mM Hepes/1% new-born calf serum) and in rotation shakes in the bottle with 1 * 10 ⁶Individual cells/ml was in 37 ℃ of incubations 2.5 hours.Then in 37 ℃ with 1 micromole Fura-2 ethoxymethyl ester (Fura-2AM; Molecular probe) handle cell 30 minutes, use sample substratum washed twice, and with 5 * 10 ⁶Individual cells/ml resuspending.Before will fluorescence spectrophotometry analyzing, under 1000rpm through centrifugal recovery cell, at the Krebs of the modification that contains sulfinpyrazone damping fluid (135mM NaCl/4.7mM KCl/1.2mM MgSO ₄/ 1.2mMKH ₂PO ₄/ 5mM NaHCO ₃/ 1mM CaCl ₂/ 2.8mM glucose/10mM hepes, pH7.4) in 1 * 10 ⁶Individual cells/ml resuspending.Bombesin is available from Sigma and Auspep.Be on the Hitachi spectrophotofluorometer (F4010) at 340nm (exciting) and 505nm (emission) with reaction times of 5nm slit width and 2 seconds and carry out fluorescence records.Adopt Grynkiewicz etc., journal of biological chemistry 260:3440-3450, the 1985 quantitative cellular calciums of describing of equation.

The present invention also provides the fat method that treats and/or prevents, this method by the host is used in conjunction with and activate the compound of receptor polypeptides of the present invention.A kind of like this compound is not Zhang, etc., nature, the disclosed ob gene product of 372:425-431 (1994).The position corresponding of receptor polypeptides of the present invention on human chromosome is in the position of the mouse chromosome of the acceptor of coding ob gene product.People ob genes encoding " full " is the factor (satiety), and this factor combination also activates receptor polypeptides of the present invention.Therefore, the compound that activates receptor polypeptides of the present invention can reduce appetite and stop fat.

Compound described above also can be used to strengthen activity level, improves the dining behavior, increases the availability of digest food and regulate accumulating of fat storage.Also can with in conjunction with and the compounds for treating illness relevant that activate receptor polypeptides of the present invention with obesity, comprise hyperlipoidemia, type ii diabetes and some cancer.

These compounds also can be used for the treatment of and/or prevent other with receptor polypeptides of the present invention or be attached to the not enough relevant illness of expression of the part on it, for example are used for stimulating neuronal growth.

The specific example that suppresses receptor polypeptides activated compound of the present invention comprises antibody, perhaps comprises oligonucleotide in some cases, thereby it is in conjunction with described acceptor but do not cause the activity that second messenger reaction stops described acceptor.

Another example is and the closely-related protein of receptors ligand, i.e. part fragment, and it has lost biological function, and not initiation reaction on being attached to acceptor the time.

Another example is the antisense constructs that adopts the antisense technology preparation.The expression that antisense technology can come controlling gene by triple helical formation or antisense DNA or RNA, these two kinds of methods are all based on the combination of polynucleotide and DNA or RNA.For example, the encode 5 ' encoding part of polynucleotide sequence of mature polypeptide of the present invention can be used for the antisense rna oligonucleotide of about 10 to 40 base pairs of design length.A kind of DNA oligonucleotide is designed to and transcribes related gene regions complementation (triple helical-referring to Lee etc., nucleic acids research, 6:3073 (1979); Cooney etc., science, 241:456, (1988); With Dervan etc., science, 251:1360 (1991)), and then stop transcribing and producing of polypeptide of the present invention.Antisense rna oligonucleotide is hybridized with mRNA in vivo, and the translation becoming of blocking-up mRNA molecule polypeptide of the present invention (antisense-Okano, J.Neurochem., 56:560 (1991); Oligodeoxynucleotide is as the antisense inhibitor (CRC press, Boca Raton, FL (1988)) of genetic expression.Oligonucleotide described above can be sent in the cell, so that can expression in vivo sense-rna and DNA, suppresses the generation of described polypeptide.

Another example is a small molecules, and these small molecules are attached on the neuropeptide receptor of the present invention, makes acceptor not stop normal biologic activity thus near its part.Micromolecular example includes but not limited to little peptide or class peptide molecule and neuropeptide tyrosine fragment and/or derivative.

The soluble form of neuropeptide receptor polypeptide of the present invention (for example, the fragment of acceptor, it is attached on the part and stops part and membrane-bound receptor interacts) also can suppress the activation of receptor polypeptides of the present invention.

The present invention also provides and will suppress the method that this compound of activated is used for the treatment of abnormal diseases relevant with the excessive activity of neuropeptide receptor polypeptide of the present invention and treatment obesity, this is because neuropeptide receptor polypeptide of the present invention can be in conjunction with neuropeptide tyrosine, the latter is the strongest known substance that causes feed behavior increase and type ii diabetes, and neuropeptide tyrosine can work in the hereditary basis of this disease.

Inhibition receptor polypeptides activated compound of the present invention can be used for the treatment of and/or preventing hypertension, and this is because neuropeptide tyrosine stimulates feritin to discharge and known neuropeptide tyrosine is relating to crown and having the potential vasoconstrictor activity during cerebral vessels.

Compound of the present invention also can be used for the treatment of Alzheimer's, this is because neuropeptide receptor ubiquity in central nervous system, and in the neurone, at this moment, they seem that memory and Alzheimer's are had regulating effect in being positioned at with preponderating.

Described compound also can be used for suppressing hippocampus the pungency transmission of neuropeptide tyrosine, and therefore can be used for the treatment of epileptic seizures, anxiety and worried.

Neuropeptide receptor ubiquity in central nervous system shows that the compound that suppresses neuropeptide receptor polypeptide of the present invention can be used as psychotolytic medicine by regulating neurotransmission.

The compound that suppresses receptor polypeptides of the present invention also can be used for the treatment of the pathologic vessels spasm, comprises crown and vasospasm cerebral vessels.

The present invention also provides and has measured the unknown part that whether can be attached on the neuropeptide receptor of the present invention and whether can be incorporated into method on it, differentiate before this method is included in and is enough to make to the part in conjunction with such acceptor is attached under the condition on the acceptor, part to be identified with comprise the neuropeptide receptor encoding sequence and also express the cells contacting of this sequence in its surface.In other embodiments, comprise the cell membrane component or the isolating free receptor of acceptor or be fixed on the combination that separation acceptor on the solid support can be used to measure part to be tested.When reconstitution cell is used for the expression of receptor purpose, preferably use to have few or do not have the active cell of endogenous receptor, so that be because due to the existence of the purpose acceptor of expressing in conjunction with (if having any bonded words).Preferred cell comprises human early stage nephrocyte, monkey kidney (HEK-293 cell), inoblast (COS), Chinese hamster ovary (CHO) cell, fruit bat or mouse L-cell.Adopt preferably that wherein to have the cell of receptor response second messenger system be host cell.Famous second messenger system is included in increase or the minimizing that the response part is attached to phosphoinositide hydrolysis, adenylate cyclase, guanylate cyclase or ion channel activity in the outer receptor domain of born of the same parents.In other embodiments, can make up the receptors bind indicator of special design.For example, fusion rotein can prepare by acceptor of the present invention is merged with the protein functional domain that receptors ligand is combined sensitivity.Here be called this functional domain self of indicator functional domain or can produce analytically detectable signal, its indication receptors ligand combination with additional molecule.

The present invention also provides the existence by detecting the coding receptor mRNA to detect the method that neuropeptide receptor polypeptide of the present invention is expressed on cell surface, this method comprises from cell and obtains total mRNA, the mRNA that so obtains contacted with nucleic acid probe (described probe comprises the nucleic acid molecule of at least 10 Nucleotide, it can be hybridized with the sequence-specific ground in the nucleic acid molecule that is included in the described acceptor of coding under hybridization conditions), detection hybridizes to the existence of the mRNA on the probe, and then detects cell to receptor expression.

The present invention also provides the method that is used to differentiate the acceptor relevant with receptor polypeptides of the present invention.Can be by differentiating these associated receptors with the homology of neuropeptide receptor polypeptide of the present invention, it is by low severity cross hybridization or by differentiating that acceptor carries out, described acceptor and relevant natural or synthetic ligands interaction or excite similar behavior after the heredity of neuropeptide receptor polypeptide of the present invention or pharmacology block.

Described gene fragment can be as the hybridization probe in cDNA library, with separation have with high homology of gene of the present invention or similar biologic activity other sequence.Such probe preferably has 50 bases or more.Described probe can be used for also differentiating with total length transcript and genomic clone or clone corresponding cDNA clone that it comprises the complete genome of the present invention that comprises adjusting and promoter region, exon and intron.The example of such screening comprises by utilizing the known dna sequence synthetic oligonucleotide probe to separate the coding region of said gene.The oligonucleotide that has with the mark of the sequence complementarity of gene of the present invention is used to screen human cDNA, genomic dna or mRNA library to determine which member of probe hybridization to the library.

Described neuropeptide receptor polypeptide and above discriminating for the compound of polypeptide can utilize by the such polypeptide of expression in vivo according to the present invention, this often is known as " gene therapy ".

Like this, for example, can external pair cell carry out the genetically engineered operation with the polynucleotide (DNA or RNA) of coded polypeptide, the patient who treats to quilt with engineering cell provides said polypeptide.Such method is well-known in the art, and also apparent by the description of this paper.For example, can use the retroviral particle pair cell of the RNA that comprises the polypeptide of the present invention of encoding to carry out the genetically engineered operation with method well known in the art.

Similarly, can carry out the genetically engineered operation by pair cell in the methods known in the art body for example, so that the expression in vivo polypeptide.As known in the art, the generation cell that produces the retroviral particle of the RNA comprise the polypeptide of the present invention of encode can be applied to the patient so that body is interior through genetically engineered manipulating cells and the said polypeptide of expression in vivo.Through description of the invention, these or other method of using polypeptide of the present invention in this way is clearly to those skilled in the art.For example, can not retrovirus through the carrier of genetically engineered manipulating cells, but adenovirus for example, it can be used in the body through the genetically engineered manipulating cells after transporting carrier combinations with suitable.

The retrovirus that can derive from above-described retrovirus plasmid vector includes but not limited to: moloneys mouse leukosis virus, spleen necrosis virus, retrovirus such as Rous sarcoma virus, Harvey sarcoma virus, avian leukosis viruses, gibbon orangutan leukosis virus, human immunodeficiency virus, adenovirus, marrow increment sarcoma virus and mammary tumor virus.In one embodiment, the retrovirus plasmid vector is from the moloneys mouse leukosis virus.

Described carrier comprises one or more promotors.Operable suitable promotor includes but not limited to: retrovirus LTR; SV 40 promotors; And human cytomegalovirus (CMV) promotor (Miller, etc., biotechnology, Vol.7, No.9,980-990 (1989) describes); Perhaps other any promotor (for example eukaryotic cell promotor, as include but not limited to histone, pol III and beta-actin promotor).Other viral promotors that uses includes but not limited to: adenovirus promoter, thymidine kinase (TK) promotor and B19 parvovirus promotor.By the description of this paper, in the ken that is chosen in those skilled in the art of suitable promotor.

The nucleotide sequence of code book invention polypeptide is under suitable promotor control.Operable suitable promotor includes, but are not limited to: adenovirus promoter (as adenovirus major late promoter); Perhaps allogeneic promoter (as cytomegalovirus (CMV) promotor); Respiratory syncytial virus (RSV) promotor; Inducible promoter (as MMT promotor, metallothionein promoter); The heat-shocked promotor; The white protein promotor; The ApoAI promotor; Human globin promoter; Viral thymidine kinase promoter (as herpes simplex thymidine kinase promoter); Retrovirus LTRs (the retrovirus LTRs that comprises above-described modification); The beta-actin promotor; With human growth hormone's promotor.Said promotor also can be the natural promoter of the gene of control coding said polypeptide.

Use retrovirus plasmid vector transduction package cell line so that form production clone.The example of packing cell that can be transfected includes but not limited to: PE501, PA317, ψ-2, ψ-AM, PA12, T19-14X, VT-19-17-H2, ψ CRE, ψ CRIP, GP+E-86, GP+envAm12 and DNA clone (Miller, human gene therapy, Vol.1, pgs.5-14 (1990) describes, and its incorporated herein by is reference in the lump).Can be by any known method in this area with described carrier transduction packing cell.These methods include but not limited to: electroporation, use liposome and CaPO ₄Precipitation.In addition, the retrovirus plasmid vector can be encapsulated in the liposome, perhaps with the lipid coupling, is administered among the host then.

Production clone produces infective retroviral vector particles, and this particle comprises the nucleotide sequence of coding said polypeptide.Can use in these retroviral vector particles bodies then or external transduction eukaryotic cell.The eukaryotic cell of transduction will be expressed the nucleotide sequence of coding said polypeptide.The eukaryotic cell that can be transduceed includes but not limited to: embryonic stem cells, embryo cancer cells and hemopoietic stem cell, liver cell, inoblast, sarcoplast, keratinocyte, endotheliocyte and bronchial epithelial cell.

Solubility neuropeptide receptor polypeptide and in conjunction with and activate acceptor of the present invention or suppress its activated compound and also can be used for making up with suitable pharmaceutical carrier.This composition comprises described solubility neuropeptide receptor polypeptide or compound and the pharmaceutically acceptable carrier or the vehicle for the treatment of significant quantity.Such carrier includes but not limited to salt solution, buffer saline, glucose, water, glycerine, ethanol and their molectron.Prescription should be suitable for mode of administration.

The present invention also provides pharmaceutical pack or test kit, and they comprise one or more containers that are filled with one or more compositions of pharmaceutical composition of the present invention.Can be in this container with the bulletin of government organs' prescribed form of managing medicine and biological products manufacturing, use or sale, this bulletin has reflected manufacturing, use or sold the mankind makes articles for use obtain the agreement of government organs.In addition, solubility neuropeptide receptor polypeptide of the present invention or compound can be treated compound with other and be used in combination.

Described pharmaceutical composition can be used in mode easily, described mode for example in part, intravenously, intraperitoneal, intramuscular, subcutaneous, the nose or the intradermal approach use.Said pharmaceutical composition is used with the significant quantity that treats and/or prevents specified disease.In general, they are used with the amount of about at least 10 micrograms/kg body weight, and in most of the cases, they are used with the amount that is no more than about 8 mg/kg body weight every day.Under most applications, consider factors such as route of administration and illness, dosage from every day about 10 microgram/kilograms to 1 mg/kg body weight.

The present invention also relates to the purposes of gene of the present invention, for example the diagnostic reagent of some disease that causes by the hereditary defect gene as diagnostic reagent.Can be by relatively dcc gene sequence and normal gene sequence detect these genes.Then, people can confirm that " sudden change " gene and abnormal receptor are active relevant.In addition, people can be at functional assays system (for example colorimetric estimation, the expression on the MacConkey flat board, complementation test, in the acceptor defect strain of HEK293 cell) in the mutant receptors gene is inserted in the suitable expression vector, it is as the another kind of method that confirms or differentiate sudden change.In case " sudden change " gene is identified, people just can screen " sudden change " acceptor gene carrier group.

Can on dna level, detect individuality with various technology with people's gene sudden change of the present invention.Can obtain diagnostic nucleic acid from patient's cell (including but not limited to for example blood, urinate that saliva is organized examination of living tissue and necrotomy material).Genomic dna can be directly used in detection, perhaps uses PCR enzymatic amplification (Saiki etc., nature, 324:163-166 (1986)) before analysis.RNA or cDNA also can be used for identical purpose.As an example, can use with the Nucleotide complementary PCR primer of code book invention polypeptide and differentiate and analyze its sudden change.For example, can by with the amplified production size of normal genotype comparison on change detect disappearance or insert.Point mutation can be through DNA and the radiolabeled RNA or the radiolabeled antisense dna sequence hybridization discriminating of amplification.Through ribonuclease A digestion, perhaps distinguish perfect paired sequence and mispairing duplex from the difference of melting temperature.This diagnostic reagent potential or or even introduction stage test in can be useful especially.

Can be by the sequence difference between direct dna sequencing method announcement reference gene and " sudden change " gene.In addition, clone's DNA section can be as the probe that detects specific DNA section.When making up with PCR, the susceptibility of this method is greatly improved.For example, use aligning primer, the single-stranded template molecule that it has double-stranded PCR product or is produced by the PCR that modifies.Finish sequencing through conventional method or with fluorescent mark through the automatic sequencing method with radiolabeled Nucleotide.

Can finish by detecting when being with or without denaturing agent on the gel change of dna fragmentation electrophoretic mobility based on the genetic test of dna sequence dna difference.Also can protect assay method (as ribonuclease protecting and S1 protection) and chemical cracking method (for example, Cotton etc., PNAS, the U.S., 85:4397-4401 (1985)) to disclose sequence variation on the specific position by nuclease.

In addition, some disease results from the variation of genetic expression or be feature with it, and this variation can be detected by the variation of mRNA.In addition, gene of the present invention can be as with reference to being used to differentiate the individuality of expressing the relevant function of the acceptor with this type that reduces.

The present invention also relates to be used for to detect the diagnostic assay method of level of change of the soluble form of various tissues neuropeptide receptor polypeptide of the present invention.The assay method that is used for detecting the soluble receptors polypeptide level of the sample that derives from the host is known those skilled in the art, comprises that radioimmunoassay, competition-combination are measured, the Western engram analysis, and preferably ELISA measures.

ELISA measures and originally comprises preparation to the specific antibody of neuropeptide receptor polypeptide antigen, preferably a kind of monoclonal antibody.In addition, preparation is at the report antibody of described monoclonal antibody.But be connected with detection reagent on the described report antibody, for example radioactivity, fluorescence or the horseradish peroxidase in this example.Then, obtain sample from the host, incubation on solid support, described upholder is the polystyrene ware for example, and it is in conjunction with the protein in the sample.By covering any free protein binding site on the ware with nonspecific proteins matter (for example bovine serum albumin) incubation.The then described monoclonal antibody of incubation, during this period, monoclonal antibody is connected on any and the neuropeptide receptor protein that the polystyrene ware is connected.Wash away all unconjugated monoclonal antibodies with damping fluid.The report antibody that will be connected to then on the horseradish peroxidase places ware, makes the report antibodies on any and antibody that neuropeptide receptor protein is connected.Wash the antibody that does not connect off.Peroxidase substrate is added in the ware.But when comparing with typical curve, the amount of the color that produces in given time bar is the proteinic amount of neuropeptide receptor that is present in the given volume patient sample.

Sequence of the present invention identifies it also is valuable to karyomit(e).This sequence specific ground target is positioned at the specific position on the one human chromosome and can hybridizes with it.In addition, the present specific site that needs to identify on the karyomit(e).At present, only there is the chromosome marking reagent of a few sequence data (repetition polymorphism) can be used for the position of marker chromosomes based on reality.DNA chromosome mapping of the present invention is the important the first step that these sequences and disease related gene are associated.

In brief, just can navigate to sequence on the karyomit(e) by prepare PCR primer (preferred 10-25bp) from cDNA.Adopt the Computer Analysis in 3 ' the untranslated zone can select primer apace, wherein primer should not crossed over above an exon on the genomic dna, otherwise makes amplification method complicated.Adopt these primers to be used for the somatic hybridization body that the PCR screening contains one human chromosome then.Have only those crossbreds that contain the people's gene corresponding just can produce amplified fragments with this primer.

The PCR of somatic hybridization body mapping is that a specific DNA is positioned quick program on the specific karyomit(e).Adopt same Oligonucleolide primers according to the present invention, usefulness comes from one group of fragment of specific karyomit(e) or big genomic clone aggregate, can realize inferior location (sublocalization) in a similar way.Can be used for similarly other mapping strategy of chromosome mapping is comprised in situ hybridization, carry out prescreen and carry out preliminary election with the karyomit(e) through airflow classification of mark, thereby construct the cDNA library of chromosome specific by hybridization.

The cDNA clone can be used for realizing the accurate chromosomal localization of single stage method with the fluorescence in situ hybridization (FISH) of Metaphase Chromosome smear.This technology can adopt the cDNA of 50 or 60 base length.Summary about this technology is consulted Verma etc., human chromosomal: basic fundamental handbook, Pergamon press, New York (1988).

In case sequence has navigated to a chromosome position accurately, then the physical location of this sequence can be associated with the genetic map data on the karyomit(e).These data for example can be at V.McKusick, finds in the human Mendelian inheritance (can by online the obtaining in the Johns Hopkins Welch of university medical science library).Relation between the disease on coming identified gene and navigate to identical chromosomal region by linkage analysis (the common heredity of the adjacent gene of physics) then.

Next need to be determined at the difference in cDNA between the affected and unaffected individuality or the genome sequence.If sudden change be observed in some or all of affected individuals, but in any one normal individual, be not observed again, this sudden change may be the cause of disease of disease so.

According to physical mapping and the present resolving power of genetic mapping technology, the cDNA that quilt accurately navigates to the chromosomal region relevant with disease can be 50-500 potential cause a disease a kind of (wherein supposition the mapping resolving power of 1 megabasse and every 20kb are arranged be a gene) in (causative) gene.

The cell of described polypeptide, its fragment or derivative or analogue or expression above-mentioned substance can be as the immunogen of producing its antibody.These antibody can be for example polyclonal antibody or monoclonal antibody.The present invention also comprises chimeric, strand and humanized antibody, and the product of Fab fragment or Fab expression library.Several different methods known in the art can be used to produce these antibody and fragment.

Can be by using the anti-antibody that produces corresponding to polypeptide of sequence of the present invention of polypeptide acquisition to animal direct injection polypeptide or to animal (preferably non-human animal).The antibodies polypeptide itself that obtains like this then.In such a way, even a fragments sequence of coded polypeptide also can be used to produce the antibody in conjunction with all natural polypeptide.With this antibody isolated polypeptide from the tissue of express polypeptide.

For MONOCLONAL ANTIBODIES SPECIFIC FOR, can use any technology that produces antibody by the continuous cell line culture that provides.Example comprises hybridoma technology (Kohler and the Milstein that produces human monoclonal antibodies, 1975, naturally, 256:495-497), trioma technology, people B-quadroma technology (Kozbor etc., 1983, current immunology, 4:72) and Epstein-Barr virus-hybridoma technology (Cole etc., 1985, monoclonal antibody and cancer therapy, Alan R.Liss company, pp.77-96).

The technology (United States Patent (USP) 4,946,778) that described relevant single-chain antibody produces can be used for producing the single-chain antibody of anti-immunogenic polypeptide product of the present invention.Also can utilize transgenic mice to express the humanized antibody of anti-immunogenic polypeptide product of the present invention.

The present invention further is illustrated with reference to the following examples; But, should understand the present invention and be not limited to these embodiment.Unless beyond doing in addition to state, all part or amounts are weight.

To understand following embodiment in order being beneficial to, now to narrate method and/or term that some often occur.

" plasmid " is by a small letter p and/or follow several capitalizations and/or numeral is named the preceding.Initial plasmid herein or can by commercial sources obtain or on unrestricted basis the public can obtain, perhaps can from available plasmid, build according to disclosed method.In addition, be known in the art for plasmid and be conspicuous those of ordinary skills with those described equivalences.

" digestion " of DNA is meant the Restriction Enzyme catalytic pyrolysis DNA that only some sequence on the DNA is worked with a kind of.The multiple Restriction Enzyme that this paper adopted can obtain by commercial sources, and its reaction conditions, cofactor and other service requirements are known to those of ordinary skills.For analysis purposes, usually the plasmid of 1 μ g or the dna fragmentation enzyme with about 2 units that are dissolved in about 20 μ l buffered soln is used.In order to separate the dna fragmentation that is used for plasmid construction, usually in a bigger volume with the DNA of enzymic digestion 5 to the 50 μ g of 20 to 250 units.At concrete Restriction Enzyme, the suitable buffered soln and the amount of substrate are stipulated by the producer.Usually adopt 37 ℃ of following about 1 hour incubation times, but this time can change according to the indication of product supplier.After digestion, reaction mixture directly carries out electrophoresis to isolate required fragment on polyacrylamide gel.

Employing is by Goeddel, D. etc., and nucleic acids research, described 8% polyacrylamide gel of 8:4057 (1980) carries out the size separation of crack fragment.

" oligonucleotide " or refer to a kind of strand poly deoxynucleosides, or refer to can be by two complementary poly deoxynucleosides chains of chemosynthesis.Therefore these synthetic oligonucleotide do not have 5 ' phosphoric acid, if not in the presence of a kind of kinases during with phosphoric acid of ATP interpolation, this oligonucleotide will can not be connected on another oligonucleotide.The synthetic oligonucleotide will be connected to not by on the dephosphorylized fragment.

" connection " be meant between two double stranded nucleic acid fragments the process that forms phosphodiester bond (Maniatis, T., etc., ibid, p.146).Unless beyond providing separately, adopt dna fragmentation to be connected 10 T4 of the unit dna ligases (" ligase enzyme ") of known damping fluid and condition, the about equimolar amount of per 0.5 μ g to realize being connected.

Except as otherwise noted, press Graham, F. and Van der Eb, A., virusology, the described method of 52:456-457 (1973) transforms.

Embodiment 1

The bacterial expression of neuropeptide receptor and purifying

Utilize the dna sequence dna (ATCC#_____) of the initial amplification coding neuropeptide receptor of PCR Oligonucleolide primers, said primer is corresponding to 5 ' and the 3 ' end sequence (cut signal peptide sequence) of finished neuropeptide receptor gene and 3 ' carrier sequence of described gene '.To join respectively in 5 ' and the 3 ' sequence corresponding to the additional nucleotide of neuropeptide receptor gene.Said 5 ' Oligonucleolide primers has sequence 5 ' CACTAAAGCTTAATGGAGCCCTCAGCCACC 3 ' (SEQ ID NO:7), and contain the HindIII restriction endonuclease sites, 18 Nucleotide of the neuropeptide receptor encoding sequence that the back then begins from the proteinic end amino acid codon of inferring of processing.Said 3 ' sequence, 5 ' ACAAGTCCTTGTCCTTCTAGAGGGC 3 ' (SEQ ID NO:8) contains the XbaI site.Said restriction endonuclease sites is corresponding to bacterial expression vector pQE-9 (Qiagen company, Chatsworth, restriction endonuclease sites CA).PQE-9 coding antibiotics resistance (Amp ^r), the replication orgin (ori) of bacterium, the adjustable promotor operon of IPTG (P/O), ribosome bind site (RBS), 6-histidine mark and restriction endonuclease sites.Then with Hind III and Xba I digestion pQE-9.The sequence of amplification is connected among the pQE-9 and is inserted in the frame that has histidine mark encoding sequence and RBS.The method of describing by (molecular clonings: laboratory manual, press of cold spring harbor laboratory, (1989)) such as Sambrook is used to connect mixture transformed into escherichia coli bacterial strain M15/rep 4 (Qiagen, companies) then.M15/rep4 comprises the multiple copied of plasmid pREP4, and this plasmid expression lacI repressor is given kalamycin resistance (Kan simultaneously ^r).Identify transformant by the energy for growth of transformant on the LB flat board, and select penbritin/kalamycin resistance bacterium colony.Separate and the affirmation plasmid DNA by restriction analysis.To contain being cloned in of required construct and replenish (O/N) growth of spending the night in the LB liquid nutrient medium of Amp (100 μ g/ml) and Kan (25 μ g/ml).With the ratio inoculation large volume culture of O/N culture with 1: 100 to 1: 250.Cell grows to optical density(OD) 600 (O.D. ⁶⁰⁰) be between 0.4 and 0.6 the time, add IPTG (sec.-propyl-B-D-thio-galactose pyran-glucoside) to ultimate density be 1mM.IPTG increases expression by deactivation lacI repressor, removing P/O induced gene.Cell was cultivated 3 to 4 hours again.Pass through centrifugal cell harvesting.Cell precipitation is dissolved chaotropic agent in the 6M Guanidinium hydrochloride.After the clarification, under the condition that the protein that allows to contain the 5-histidine mark is combined closely, on nickel-NAT resin by chromatography purifying dissolved neuropeptide receptor (chromatography is learned magazine 411:177-184 (1984) for Hochuli, E. etc.) from solution.Neuropeptide receptor protein (85% purity) is eluted from post with 6M Guanidinium hydrochloride (pH value 5.0),, transfer to 3M Guanidinium hydrochloride, 100mM phosphoric acid salt sodium, 10mM gsh (reduced form), 2mM gsh (oxidized form) for renaturation., after 12 hours protein is dialysed to the 10mM sodium phosphate in this solution incubation.

Embodiment 2

Express recombinant neuropeptide receptor in the COS cell

The plasmid expression of neuropeptide receptor-HA derives from carrier pcDNA3/Amp (Invitrogen), it comprises: 1) SV 40 replication orgin, 2) ampicillin resistance gene, 3) colibacillary replication orgin), 4) CMV promotor is thereafter polylinker district, SV 40 introns and polyadenylation site.With the dna fragmentation of the complete neuropeptide receptor precursor of coding with in frame, be integrated into the polylinker district of 3 ' terminal HA marker clone to said carrier, thus, being expressed under the control of CMV promotor of recombinant protein.The HA mark is corresponding to from the proteinic epi-position of influenza hemagglutinin, and this point was described (I.Wilson, H.Niman, R.Heighten, A Cherenson, M.Connolly, and R.Lerner, 1984, cell 37:767) in the past.The proteic fusion of HA and target cell makes the recombinant protein that has antibody (its identification HA epi-position) be easy to detect.

The plasmid construction strategy is described below:

With the dna sequence dna (ATCC#_____) of two kinds of primers through PCR structure coding neuropeptide receptor, said primer is: 5 ' primer, 5 ' CCTAGGATGCCCCTCTGCTGCAGCGG 3 ' (SEQ ID NO:9) comprises the BamHI site; 3 ' primer, 5 ' ACAAGTCCTTGTCCTTCTGAGGGC 3 ' (SEQ ID NO:10) comprises the sequence of last 17 Nucleotide (not comprising terminator codon) that are complementary to XbaI site, translation stop codon and neuropeptide receptor encoding sequence district.The PCR product comprises BamH I site, encoding sequence, translation stop codon and XbaI site thus.Dna fragmentation with BamHI and XbaI digestion pcr amplification also is connected with carrier pcDNA3/Amp.To connect mixture and be transformed among the coli strain SURE (can be from the Stratagene cloning system, La Jolla obtains), the culture that transforms will be seeded on the ampicillin medium flat board, and the screening resistance clone.Isolated plasmid dna from transformant, and detect whether there is correct fragment with restriction analysis.For the express recombinant neuropeptide receptor, by DEAE-DEXTRAN method expression vector rotaring redyeing COS cell (J.Sambrook, E.Fritsch, T.Maniatis, molecular cloning: laboratory manual, cold spring harbor laboratory's publication, (1989)).Detect the proteic expression of neuropeptide receptor HA (E.Harlow, D.Lane, antibody: laboratory manual, cold spring harbor laboratory's publication, (1988)) by radio-labeled and immuno-precipitation.With the two day usefulness of cell after transfection ¹⁵S-halfcystine mark 8 hours.Collect culture then and use washing agent lysing cell (RIPA damping fluid (150mM NaCl, 1%NP-40,0.1%SDS, 1%NP-40,0.5%DOC, 50mM Tris, pH7.5) (Wilson, I. etc., Id.37:767 (1984)).Monoclonal antibody specific sedimentation cell lysate and culture with HA.Analysing protein precipitation on the 15%SDS-PAGE gel.

Embodiment 3

Utilize the baculovirus expression system clone and express neuropeptide receptor

Utilize PCR Oligonucleolide primers amplification coding total length neuropeptide receptor protein DNA sequence (ATCC#_____), said primer is corresponding to 5 ' and 3 ' sequence of this gene:

Neuropeptide receptor 5 ' primer has sequence 5 ' CGGGATCCGCCATC ATGGAGCCCTCAGCCACC 3 ' (SEQ ID NO:11), comprise BamHI restriction site (runic), next be similar eukaryotic cell translation initiation useful signal (Kozak, M., J.Mol.Biol., 196:947-950 (1987)) 6 Nucleotide, they are just after 18 Nucleotide of described gene (translation initiation codon " ATG " has underscore).

3 ' primer has sequence 5 ' ACAAGTCCTTGTCCTTCTAGAGGGC 3 ' (SEQID NO:12), comprises the cleavage site and 5 Nucleotide that are complementary to neuropeptide receptor gene 3 '-non-translated sequence of restriction enzyme XbaI.(" Geneclean " BIO101 company, La Jolla Ca.) separate the sequence that increases from 1% sepharose with the commercial reagent box.Then with said fragment with corresponding nucleic acids restriction endonuclease BamHI and XbaI digestion, and as purifying as described in the embodiment 1.This fragment is appointed as F2.

Carrier pA2 (is formed by the pVL941 carrier modification, discussion sees below) be used for marking protein, baculovirus expression system is used in this expression, and (summary is referring to Summer, M.D. and Smith, G.E.1987, baculovirus vector and insect cell cultural method handbook, testing station's communique 1555 of Texas's agricultural).This expression vector comprises the strong polyhedrin promotor of the polygonal virus of autographa california multinuclear type (AcMNPV), and its back is the recognition site of restriction endonuclease BamHI and X baI.The polyadenylation site of monkey disease poison (SV) 40 is used for effective polyadenylation.In order easily to select recombinant virus, colibacillary beta-galactosidase gene is inserted with same direction as the polyhedrin promotor, be thereafter the polyadenylation signal of polyhedron gene.The both sides of polyhedrin sequence are the virus sequences of homologous recombination that is used for the wild-type virus DNA of cell-mediated cotransfection.Can use much other baculovirus vector replacement A2, and said carrier such as pGR1, pAc373, pVL941 and pAcIM1 (Luckow, V.A. and Summer, M.D., virusology, 170:31-39).

Digest said plasmid with restriction enzyme BamHI and XbaI, and utilize calf intestinal phosphatase enzyme to make the plasmid dephosphorylation with methods known in the art.Then as the description of embodiment 1 from 1% sepharose DNA isolation.This carrier DNA is appointed as V2.

Connect F2 fragment and said dephosphorylated plasmid V2 with the T4 dna ligase.Transform DH5 α then, and utilize restriction enzyme BamHI and XbaI to differentiate the bacterium that contains said plasmid (pBac neuropeptide receptor) with neuropeptide receptor gene.Confirm the fragments sequence cloned by dna sequencing.

With lipofection (Felgner etc., Proc. Natl. Acad. Sci.USA, 84:7413-7417 (1987)) with 5 μ g plasmid pBac neuropeptide receptors and commercially available the linearized baculovirus (" BaculoGold of 1.0 μ g ^TMBaculovirus DNA ", Pharmingen, San Diego, CA.) cotransfection.

With 1 μ g BaculoGold ^TM(Life Technologies company, Gaithersburg mix in the aseptic hole of droplet plate MD) containing 50 microlitre serum-free Grace ' s substratum for viral DNA and 5 μ g plasmid pBac neuropeptide receptors.After adding 10 microlitre lipofectin reagents and 90 microlitre Grace ' s substratum, mix to be incorporated under the room temperature and cultivated 15 minutes.Then transfection mixture is dropwise added in the Sf9 insect cell (ATCC CRL1711), said cell inoculation is on the 35mm tissue culture plate with 1ml serum-free Grace ' s substratum.The waggle flat board is to mix the solution of new interpolation.Then flat board was cultivated 5 hours down at 27 ℃.After 5 hours, remove transfection solution, add 1 milliliter of Grace ' s insect substratum that is supplemented with 10% foetal calf serum from flat board.Flat board is put back into incubator, 27 ℃ of following cultured continuously 4 days.

After 4 days, collect supernatant liquor, carry out plaque measurement with similar Summers and the described method of Smith (the same).A bit change sepharose (the Life Technologies company that is to use with " Blue Gal ", Gaithersburg), its make be easy to separate dye blue plaque (the detailed description of " plaque measurement " also can the insect cell of Life Technologies company (Gaithersburg) issue cultivate and baculovirus users' guidebook 9-10 page or leaf in find).

After four days, the virus of serial dilution is added in the cell, dye blue plaque with Eppendorf pipette tip picking.The agar that will comprise recombinant virus then is suspended in the Eppendcrf pipe that comprises 200 microlitre Grace ' s substratum.Through the brief centrifugal agar of removing, the supernatant liquor that will comprise recombinant baculovirus is used for infecting the Sf9 cell that is inoculated into 35 millimeters culture dish.After 4 days, gather in the crops the supernatant liquor in these culture dish, in 4 ℃ of storages.

The Sf9 cell is grown in the Grace ' substratum that is supplemented with 10% hot deactivation FBS.Infection multiplicity (MOI) 2 times with recombinant baculovirus V-neuropeptide receptor cells infected.After 6 hours, remove substratum, (LifeTechnologies company Gaithersburg) substitutes with SF900II substratum (subtracting methionine(Met) and halfcystine).After 42 hours, add 5 μ Ci ³⁵S methionine(Met) and 5 μ Ci ³⁵S halfcystine (Amersham).Before centrifugal results, cell further to be cultivated 16 hours, labelled protein demonstrates through SDS-PAGE and radioautograph.

Embodiment 4

The fibrocyte that is expressed as via gene therapy obtains from a research object by Skin biopsy.Be placed on the tissue that obtains on the tissue culture medium (TCM) and be divided into fritter.Small tissue blocks is placed on the wet surface of tissue culture flasks, wherein places about 10 block organizations in every bottle.Bottle is placed upside down, and lid is tight and spend the night under room temperature.At room temperature place counter-rotating bottle after 24 hours, tissue block still is fixed on the bottle bottom, adds fresh culture (for example containing 10%FBS, Ham ' the s F12 substratum of penicillin and Streptomycin sulphate), then in 37 ℃ of about 1 weeks of following incubation.At this moment add fresh culture, changed a subculture subsequently every several days.After cultivating for 2 weeks again, an individual layer inoblast has appearred.With monolayer cell through tryptic digestion and put into bigger bottle.

With EcoRI and Hind III digestion side the long terminal repetition pMV-7 (Kirschmeier, P.T. etc., DNA, 7:219-25 (1988)) of Moloney murine sarcoma virus is arranged, next handle with the calf intestinal phosphatase enzyme.Linear carrier fractional separation and use granulated glass sphere purifying in addition on sepharose.

Adopt the cDNA of corresponding with 5 ' and 3 ' end sequence respectively PCR primer amplification code book invention polypeptide.5 ' primer comprises an EcoRI site, and 3 ' primer contains a Hind III site.In the presence of the T4 dna ligase, the EcoRI of the linearizing skeleton of the Moloney murine sarcoma virus of equivalent and amplification added with Hind III fragment be in the same place, be suitable for keeping resulting mixture under the condition that two fragments connect.Should connect mixture and be used for transform bacteria HB101, in order to confirm this carrier to have the correct gene of interest of insertion bacterium was coated on the agar plate that contains kantlex then.

Amphophilic (amphotropic) pA317 or GP+am12 packing cell are cultivated in the tissue culturing plate of Dulbecco ' the s improvement Eagles substratum (DMEM) that contains 10% calf serum (CS), penicillin and Streptomycin sulphate, be paved with density until reaching.The carrier that will contain described gene then adds in the substratum also with this carrier transduction packing cell.Packing cell is produced immediately and is contained the infective virion of having of this gene (packing cell is known as and produces cell now).

In the production cell of transduction, add fresh substratum, next be paved with the 10cm flat board of producing cell and collect substratum from one.The substratum that contains infectious viral particle filters to remove the production cell of desorption (detached) through millipore filter, utilizes this substratum to go to infect inoblast then.Be paved with from fibroblastic Asia to remove substratum the flat board and promptly to replace and come from the substratum of producing cell.Remove this substratum and replace fresh substratum.If the titre of virus is very high, all so in fact inoblasts are all infected and need not to select.If titre is very low, so need to adopt have as NeoOr HisThe retrovirus of alternative mark like this.

Inoblast with through engineering approaches is injected into the host then, it or separately injection or on cytodex 3 microcarrier beads, grown to inject again after being paved with.This moment, inoblast produced protein.

According to above instruction, many improvement of the present invention and variation all are possible, therefore can other mode implement the present invention within the scope of the appended claims.

Sequence table (1) general information: (i) applicant: LI etc. are denomination of invention (ii): human neuropeptide receptor is sequence number (iii): 12 (iv) addresses:

(A) addressee: CARELLA, BYRNE, BAIN, GILFILLAN,

CECCHI, STEWART and OLSTEIN

(B) street: 6 BECKER FARM ROAD

(C) city: ROSELAND

(D) state: New Jersey

(E) country: the U.S.

(F) postcode: 07068 (v) computer-reader form:

(A) media type: 3.5 inches disks

(B) computer: IBM PS/2

(c) operating system: MS-DOS

(D) software: WORD PERFECT 5.1 (vi) current request for data:

(A) application number:

(B) applying date:

(C) classification number: (viii) lawyer/proxy's information:

(A) name: FERRARO, GREGORY D.

(B) registration number: 36,134

(c) case number/number of documents: 325800-268 (ix) telecom information:

(A) phone: 201-994-1700

(B) fax: the information of 201-994-1744 (2) SEQ ID NO:1: (i) sequence signature:

(A) length: 1209 base pairs

(B) type: nucleic acid

(C) chain: strand

( D ) ： ( ii ) ：cDNA ( xi ) ：SEQ ID NO：1ATGGAGCCCT CAGCCACCCC AGGGGCCCAG ATGGGGGTCC CCCCTGGCAG CAGACAGCCG 60TCCCCTGTGC CTCCAGACTA TGAAGATGAG TTTCTCCGCT ATCTGTGGCG TGATTATCTG 120TACCCAAAAC AGTATGAGTG GGTCCTCATC CCAGCCTATG TGGCTGTGTT CGTCGTGGCC 180CTGGTGGGCA ACACGCTGGT CTGCCTGGCC GTGTGGCGGA ACCACCACAT GAGGACAGTC 240ACCAACTACT TCATTGTCAA CCTGTCCCTG GCTGACGTTC TGGTGACTGC TATCTGCCTG 300CCGGCCAGCC TGCTGGTGGA CATCACTGAG TCCTGGCTGT TCGGCCATGC CCTCTGCAAG 360GTCATCCCCT ATCTACAGGC TGTGTCCGTG TCAGTGGCAG TGCTAACTCT CAGCTTCATC 420GCCCTGGACC GCTGGTATGC CATCTGCCAC CCACTATTGT TCAAGAGCAC AGCCCGGCGG 480GCCCGTGGCT CCATCCTGGG CATCTGGGCT GTGTCGCTGG CCATCATGGT GCCCCAGGCT 540GCAGTCATGG AATGCAGCAG TGTGCTGCCT GAGCTAGCCA ACCGCACACG GCTCTTCTCA 600GTCTGTCATG AACGCTGGGC AGATGACCTC TATCCCAAGA TCTACCACAG TTGCTTCTTT 660ATTGTCACCT ACCTGGCCCC ACTGGGCCTC ATGGCCATGG CCTATTTCCA GATATTCCGC 720AACCTCTGGG GCCGCCAGAT CCCCGGCACC ACCTCAGCAC TGGTGCGGAA CTGGAAGCGC 780CCCTCAGACC AGCTGGGGGA CCTGGAGCAG GGCCTGAGTG GAGAGCCCCA GCCCCGGGGC 840CGCGCCTTCC TGGCTGAAGT GAAGCAGATG CGTGCACGGA GGAAGACAGC CAAGATGCTG 900ATGGTGGTGC TGCTGGTCTT CGCCCTCTGC TACCTGCCCA TCAGCGTCCT CAATGTCCTT 960AAGAGGGTGT TCGGGATGTT CCGCCAAGCC AGTGACCGCG AAGCTGTCTA CGCCTGCTTC 1020ACCTTCTCCC ACTGGCTGGT GTACGCCAAC AGCGCTGCCA ACCCCATCAT CTACAACTTC 1080CTCAGTGGCA AATTCCGGGA GCAGTTTAAG GCTGCCTTCT CCTGCTGCCT GCCTGGCCTG 1140GGTCCCTGCG GCTCTCTGAA GGCCCCTAGT CCCCGCTCCT CTGCCAGCCA CAAGTCCTTG 1200TCCTTGTAG 1209 ( 2 ) SEQ ID NO：2： ( i ) ：

(A) length: 402 amino acid

(B) type: amino acid

(C) chain:

(D) topological framework: linearity is molecule type (ii): protein (xi) sequence description: SEQ ID NO:2:Met Glu Pro Ser Ala Thr Pro Gly Ala Gln Met Gly Val Pro Pro

5 10 15Gly?Ser?Arg?Glu?Pro?Ser?Pro?Val?Pro?Pro?Asp?Tyr?Glu?Asp?Glu

20 25 30Phe?Leu?Arg?Tyr?Leu?Trp?Arg?Asp?Tyr?Leu?Tyr?Pro?Lys?Gln?Tyr

35 40 45Glu?Trp?Val?Leu?Ile?Ala?Ala?Tyr?Val?Ala?Val?Phe?Val?Val?Ala

50 55 60Leu?Val?Gly?Asn?Thr?Leu?Val?Cys?Leu?Ala?Val?Trp?Arg?Asn?His

65 70 75His?Met?Arg?Thr?Val?Thr?Asn?Tyr?Phe?Ile?Val?Asn?Leu?Ser?Leu

80 85 90Ala?Asp?Val?Leu?Val?Thr?Ala?Ile?Cys?Leu?Pro?Ala?Ser?Leu?Leu

95 100 105Val?Asp?Ile?Thr?Glu?Ser?Trp?Leu?Phe?Gly?His?Ala?Leu?Cys?Lys

110 115 120Val?Ile?Pro?Tyr?Leu?Gln?Ala?Val?Ser?Val?Ser?Val?Ala?Val?Leu

125 130 135Thr?Leu?Ser?Phe?Ile?Ala?Leu?Asp?Arg?Trp?Tyr?Ala?Ile?Cys?His

140 145 150Pro?Leu?Leu?Phe?Lys?Ser?Thr?Ala?Arg?Arg?Ala?Arg?Gly?Ser?Ile

155 160 165Leu?Gly?Ile?Trp?Ala?Val?Ser?Leu?Ala?Ile?Met?Val?Pro?Gln?Ala

170 175 180Ala?Val?Met?Glu?Cys?Ser?Ser?Val?Leu?Pro?Glu?Leu?Ala?Asn?Arg

185 190 195Thr?Arg?Leu?Phe?Ser?Val?Cys?Asp?Glu?Arg?Trp?Ala?Asp?Asp?Leu

200 205 210Tyr?Pro?Lys?Ile?Tyr?His?Ser?Cys?Phe?Phe?Ile?Val?Thr?Tyr?Leu

215 220 225Ala?Pro?Leu?Gly?Leu?Met?Ala?Met?Ala?Tyr?Phe?Gln?Ile?Phe?Arg

230 235 240Lys?Leu?Trp?Gly?Arg?Gln?Ile?Pro?Gly?Thr?Thr?Ser?Ala?Leu?Val

245 250 255Arg?Asn?Trp?Lys?Arg?Pro?Ser?Asp?Gln?Leu?Gly?Asp?Leu?Glu?Gln

260 265 270Gly?Leu?Ser?Gly?Glu?Pro?Gln?Pro?Arg?Gly?Arg?Ala?Phe?Leu?Ala

275 280 285Glu?Val?Lys?Gln?Met?Arg?Ala?Arg?Arg?Lys?Thr?Ala?Lys?Met?Leu

290 295 300Met?Val?Val?Leu?Leu?Val?Phe?Ala?Leu?Cys?Tyr?Leu?Pro?Ile?Ser

305 310 315Val?Leu?Asn?Val?Leu?Lys?Arg?Val?Phe?Gly?Met?Phe?Arg?Gln?Ala

320 325 330Ser?Asp?Arg?Glu?Ala?Val?Tyr?Ala?Cys?Phe?Thr?Phe?ser?His?Trp

335 340 345Leu?Val?Tyr?Ala?Asn?Ser?Ala?Ala?Asn?Pro?Ile?Ile?Tyr?Asn?Phe

350 355 360Leu?Ser?Gly?Lys?Phe?Arg?Glu?Gln?Phe?Lys?Ala?Ala?Phe?Ser?Cys

365 370 375Cys?Leu?Pro?Gly?Leu?Gly?Pro?Cys?Gly?Ser?Leu?Lys?Ala?Pro?Ser

380 385 390Pro?Arg?Ser?Ser?Ala?Ser?His?Lys?Ser?Leu?Ser?Leu

The information of 395 400 (2) SEQ ID NO:3: (i) sequence signature:

(A) length: 1110 base pairs

(B) type: nucleic acid

(C) chain: strand

( D ) ： ( ii ) ：cDNA ( xi ) ：SEQ ID NO：3：ATGGAGCCCT CAGCCACCCC AGGGGCCCAG ATGGGGGTCC CCCCTGGCAG CAGAGAGCCG 60TCCCCTGTGC CTCCAGACTA TGAAGATGAG TTTCTCCGCT ATCTGTGGCG TGATTATCTG 120TACCCAAAAC AGTATGAGTG GGTCCTCATC GCAGCCTATG TGGCTGTGTT CGTCGTGGCC 180CTGGTGGGCA ACACGCTGGT CTGCCTGGCC GTGTGGCGGA ACCACCACAT GAGGACAGTC 240ACCAACTACT TCATTGTCAA CCTGTCCCTG GCTGACGTTC TGGTGACTGC TATCTGCCTG 300CCGGCCAGCC TGCTGGTGGA CATCACTGAG TCCTGGCTGT TCGGCCATGC CCTCTGCAAG 360GTCATCCCCT ATCTACAGGC TGTGTCCGTG TCAGTGGCAG TGCTAACTCT CAGCTTCATC 420CCCCTGGACC GCTGGTATGC CATCTGCCAC CCACTATTGT TCAAGAGCAC AGCCCGGCGG 480GCCCGTGGCT CCATCCTGGG CATCTGGGCT GTGTCGCTGG CCATCATGGT GCCCCAGGCT 540GCAGTCATGC AATCCAGCAG TGTGCTGCCT GAGCTAGCCA ACCGCACACG GCTCTTCTCA 600CTCTGTCATG AACGCTGGGC AGATGACCTC TATCCCAAGA TCTACCACAG TTGCTTCTTT 660ATTGTCACCT ACCTGGCCCC ACTGGGCCTC ATGGCCATGG CCTATTTCCA GATATTCCGC 720AAGCTCTGGG GCCGCCAGAT CCCCGGCACC ACCTCAGCAC TGGTGCGGAA CTGGAAGCGC 780CCCTCAGACC AGCTGGGGGA CCTGGAGCAG GGCCTGAGTG GAGAGCCCCA GCCCCGGGGC 840CGCGCCTTCC TGGCTGAAGT GAAGCAGATG CGTGCACGGA GGAAGACAGC CAAGATGCTG 900ATGGTGGTGC TGCTGGTCTT CGCCCTCTGC TACCTCCCCA TCAGCGTCCT CAATGTCCTT 960AAGAGGGTGT TCGGGATGTT CCGCCAAGCC AGTGACCGCG AAGCTGTCTA CGCCTGCTTC 1020ACCTTCTCCC ACTGGCTGGT GTACGCCAAC AGCGCTGCCA ACCCCATCAT CTACAACTTC 1080CTCAGTGGCC TTCCCTGGAG TCTGCTCTAA 1110 ( 2 ) SEQ ID NO：4： ( i ) ：

(A) length: 369 base pairs

(B) type: amino acid

(C) chain: strand

(D) topological framework: linearity is molecule type (ii): cDNA (xi) sequence description: SEQ ID NO:4:Met Glu Pro Ser Ala Thr Pro Gly Ala Gln Met Gly Val Pro Pro

5 10 15Gly?Ser?Arg?Glu?Pro?Ser?Pro?Val?Pro?Pro?Asp?Tyr?Glu?Asp?Glu

20 25 30Phe?Leu?Arg?Tyr?Leu?Trp?Arg?Asp?Tyr?Leu?Tyr?Pro?Lys?Gln?Tyr

35 40 45Glu?Trp?Val?Leu?Ile?Ala?Ala?Tyr?Val?Ala?Val?Phe?Val?Val?Ala

50 55 60Leu?Val?Gly?Asn?Thr?Leu?Val?Cys?Leu?Ala?Val?Trp?Arg?Asn?His

65 70 75His?Met?Arg?Thr?Val?Thr?Asn?Tyr?Phe?Ile?Val?Asn?Leu?Ser?Leu

80 85 90Ala?Asp?Val?Leu?Val?Thr?Ala?Ile?Cys?Leu?Pro?Ala?Ser?Leu?Leu

95 100 105Val?Asp?Ile?Thr?Glu?Ser?Trp?Leu?Phe?Gly?His?Ala?Leu?Cys?Lys

110 115 120Val?Ile?Pro?Tyr?Leu?Gln?Ala?Val?Ser?Val?Ser?Val?Ala?Val?Leu

125 130 135Thr?Leu?Ser?Phe?Ile?Ala?Leu?Asp?Arg?Trp?Tyr?Ala?Ile?Cys?His

140 145 150Pro?Leu?Leu?Phe?Lys?Ser?Thr?Ala?Arg?Arg?Ala?Arg?Gly?Ser?Ile

155 160 165Leu?Gly?Ile?Trp?Ala?Val?Ser?Leu?Ala?Ile?Met?Val?Pro?Gln?Ala

170 175 180Ala?Val?Met?Glu?Cys?Ser?Ser?Val?Leu?Pro?Glu?Leu?Ala?Asn?Arg

185 190 195Thr?Arg?Leu?Phe?Ser?Val?Cys?Asp?Glu?Arg?Trp?Ala?Asp?Asp?Leu

200 205 210Tyr?Pro?Lys?Ile?Tyr?His?Ser?Cys?Phe?Phe?Ile?Val?Thr?Tyr?Leu

215 220 225Ala?Pro?Leu?Gly?Leu?Met?Ala?Met?Ala?Tyr?Phe?Gln?Ile?Phe?Arg

230 235 240Lys?Leu?Trp?Gly?Arg?Gln?Ile?Pro?Gly?Thr?Thr?Ser?Ala?Leu?Val

245 250 255Arg?Asn?Trp?Lys?Arg?Pro?Ser?Asp?Gln?Leu?Gly?Asp?Leu?Glu?Gln

260 265 270Gly?Leu?Ser?Gly?Glu?Pro?Gln?Pro?Arg?Gly?Arg?Ala?Phe?Leu?Ala

275 280 285Glu?Val?Lys?Gln?Met?Arg?Ala?Arg?Arg?Lys?Thr?Ala?Lys?Met?Leu

290 295 300Met?Val?Val?Leu?Leu?Val?Phe?Ala?Leu?Cys?Tyr?Leu?Pro?Ile?Ser

305 310 315Val?Leu?Asn?Val?Leu?Lys?Arg?Val?Phe?Gly?Met?Phe?Arg?Gln?Ala

320 325 330Ser?Asp?Arg?Glu?Ala?Val?Tyr?Ala?Cys?Phe?Thr?Phe?Ser?His?Trp

335 340 345Leu?Val?Tyr?Ala?Asn?Ser?Ala?Ala?Asn?Pro?Ile?Ile?Tyr?Asn?Phe

350 355 360Leu?Ser?Gly?Leu?Pro?Trp?Ser?Leu?Leu

The information of 365 (2) SEQ ID NO:5: (i) sequence signature:

(A) length: 1133 base pairs

(B) type: nucleic acid

(C) chain: strand

( D ) ： ( ii ) ：cDNA ( xi ) ：SEQ ID NO：5：ATGGAGCCCT CAGCCACCCC AGGGGCCCAG ATGGGGGTCC CCCCTGGCAG CAGAGACCCC 60TCCCCTGTGC CTCCAGACTA TGAAGATGAG TTTCTCCGCT ATCTGTGGCG TGATTATCTG 120TACCCAAAAC AGTATGAGTG GGTCCTCATC GCAGCCTATG TGGCTGTGTT CGTCGTGGCC 180CTGGTGGGCA ACACGCTGGT CTGCCTGGCC GTGTGGCGGA ACCACCACAT GAGGACAGTC 240ACCAACTACT TCATTGTCAA CCTGTCCCTG GCTGACGTTC TGGTGACTGC TATCTGCCTG 300CCGGCCAGCC TGCTGGTGGA CATCACTGAG TCCTGGCTGT TCGGCCATGC CCTCTGCAAG 360GTCATCCCCT ATCTACAGGC TGTGTCCGTG TCAGTGGCAG TGCTAACTCT CAGCTTCATC 420GCCCTGGACC GCTGGTATGC CATCTGCCAC CCACTATTGT TCAAGAGCAC AGCCCGGCGG 480GCCCGTGGCT CCATCCTGGG CATCTGGGCT GTGTCGCTGG CCATCATGGT GCCCCAGGCT 540GCAGTCATGG AATGCAGCAG TGTGCTGCCT GAGCTAGCCA ACCGCACACG GCTCTTCTCA 600GTCTGTGATG AACGCTGGGC AGATGACCTC TATCCCAAGA TCTACCACAG TTGCTTCTTT 660ATTGTCACCT ACCTGGCCCC ACTGGGCCTC ATGGCCATGG CCTATTTCCA GATATTCCGC 720AAGCTCTGGG GCCGCCAGAT CCCCGGCACC ACCTCAGCAC TGGTGCGGAA CTGGAAGCGC 780CCCTCAGACC AGCTGGGGGA CCTGGAGCAG GGCCTGAGTG GAGAGCCCCA GCCCCGGGGC 840CGCGCCTTCC TGGCTGAAGT GAAGCAGATG CGTGCACGGA GGAAGACAGC CAAGATGCTG 900ATGGTGGTGC TGCTGGTCTT CGCCCTCTGC TACCTGCCCA TCAGCGTCCT CAATGTCCTT 960AAGAGGGTGT TCGGGATGTT CCGCCAAGCC AGTGACCGCG AAGCTGTCTA CGCCTGCTTC 1020ACCTTCTCCC ACTGGCTGGT GTACGCCAAC AGCGCTGCCA ACCCCATCAT CTACAACTTC 1080CTCAGTGGAT GTAAAGAGAA GAGTCTAGTT CTGTCCTGAC CATCGTGCCC CGG 1133 ( 2 ) SEQ ID NO：6：

(i) sequence signature:

(A) length: 377 amino acid

(B) type: amino acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: cDNA

(xi) sequence description: SEQ ID NO:6:Met Glu Pro Ser Ala Thr Pro Gly Ala Gln Met Gly Val Pro Pro

5 10 15Gly?Ser?Arg?Glu?Pro?Ser?Pro?Val?Pro?Pro?Asp?Tyr?Glu?Asp?Glu

20 25 30Phe?Leu?Arg?Tyr?Leu?Trp?Arg?Asp?Tyr?Leu?Tyr?Pro?Lys?Gln?Tyr

35 40 45Glu?Trp?Val?Leu?Ile?Ala?Ala?Tyr?Val?Ala?Val?Phe?Val?Val?Ala

50 55 60Leu?Val?Gly?Asn?Thr?Leu?Val?Cys?Leu?Ala?Val?Trp?Arg?Asn?His

65 70 75His?Met?Arg?Thr?Val?Thr?Asn?Tyr?Phe?Ile?Val?Asn?Leu?Ser?Leu

80 85 90Ala?Asp?Val?Leu?Val?Thr?Ala?Ile?Cys?Leu?Pro?Ala?Ser?Leu?Leu

95 100 105Val?Asp?Ile?Thr?Glu?Ser?Trp?Leu?Phe?Gly?His?Ala?Leu?Cys?Lys

110 115 120Val?Ile?Pro?Tyr?Leu?Gln?Ala?Val?Ser?Val?Ser?Val?Ala?Val?Leu

125 130 135Thr?Leu?Ser?Phe?Ile?Ala?Leu?Asp?Arg?Trp?Tyr?Ala?Ile?Cys?His

140 145 150Pro?Leu?Leu?Phe?Lys?Ser?Thr?Ala?Arg?Arg?Ala?Arg?Gly?Ser?Ile

155 160 165Leu?Gly?Ile?Trp?Ala?Val?Ser?Leu?Ala?Ile?Met?Val?Pro?Gln?Ala

170 175 180Ala?Val?Met?Glu?Cys?Ser?Ser?Val?Leu?Pro?Glu?Leu?Ala?Asn?Arg

185 190 195Thr?Arg?Leu?Phe?Ser?Val?Cys?Asp?Glu?Arg?Trp?Ala?Asp?Asp?Leu

200 205 210Tyr?Pro?Lys?Ile?Tyr?His?Ser?Cys?Phe?Phe?Ile?Val?Thr?Tyr?Leu

215 220 225Ala?Pro?Leu?Gly?Leu?Met?Ala?Met?Ala?Tyr?Phe?Gln?Ile?Phe?Arg

230 235 240Lys?Leu?Trp?Gly?Arg?Gln?Ile?Pro?Gly?Thr?Thr?Ser?Ala?Leu?Val

245 250 255Arg?Asn?Trp?Lys?Arg?Pro?Ser?Asp?Gln?Leu?Gly?Asp?Leu?Glu?Gln

260 265 270Gly?Leu?Ser?Gly?Glu?Pro?Gln?Pro?Arg?Gly?Arg?Ala?Phe?Leu?Ala

275 280 285Glu?Val?Lys?Gln?Met?Arg?Ala?Arg?Arg?Lys?Thr?Ala?Lys?Met?Leu

290 295 300Met?Val?Val?Leu?Leu?Val?Phe?Ala?Leu?Cys?Tyr?Leu?Pro?Ile?Ser

305 310 315Val?Leu?Asn?Val?Leu?Lys?Arg?Val?Phe?Gly?Met?Phe?Arg?Gln?Ala

320 325 330Ser?Asp?Arg?Glu?Ala?Val?Tyr?Ala?Cys?Phe?Thr?Phe?Ser?His?Trp

335 340 345Leu?Val?Tyr?Ala?Asn?Ser?Ala?Ala?Asn?Pro?Ile?Ile?Tyr?Asn?Phe

350 355 360Leu?Ser?Gly?Cys?Lys?Glu?Lys?Ser?Leu?Val?Leu?Ser?Pro?Ser?Cys

The information of 365 370 375Pro Gly (2) SEQ ID NO:7:

(i) sequence signature:

(A) length: 30 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: oligonucleotide

(xi) sequence description: SEQ ID NO:7:CACTAAAGCT TAATGGAGCC CTCAGCCACC 30

(2) information of SEQ ID NO:8:

(i) sequence signature:

(A) length: 26 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: oligonucleotide

(xi) sequence description: SEQ ID NO:8:ACAAGTCCTT GTCCTTCTAG AGGGC 26

(2) information of SEQ ID NO:9:

(i) sequence signature:

(A) length: 26 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: oligonucleotide

(xi) sequence description: SEQ ID NO:9:CCTAGGATGC CCCTCTGCTG CAGCGG 26

(2) information of SEQ ID NO:10:

(i) sequence signature:

(A) length: 25 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: oligonucleotide

(xi) sequence description: SEQ ID NO:10:ACAAGTCCTT GTCCTTCTAG AGGGC 25

(2) information of SEQ ID NO:11:

(i) sequence signature:

(A) length: 32 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: oligonucleotide

(xi) sequence description: SEQ ID NO:11:CGGGATCCGC CATCATGGAG CCCTCAGCCA CC 32

(2) information of SEQ ID NO:12:

(i) sequence signature:

(A) length: 25 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: oligonucleotide

(xi) sequence description: SEQ ID NO:12:ACAAGTCCTT GTCCTTCTAG AGGGC 25

Claims (33)

1. isolating polynucleotide, this polynucleotide comprise the member who is selected from as next group:

(a) a kind of polynucleotide, the polypeptide shown in this polynucleotide encoding SEQ ID NO:2;

(b) a kind of polynucleotide, this polynucleotide can with the multi-nucleotide hybrid of (a), and and they have at least 70% homogeny;

(c) polynucleotide passage of a kind of (a) or polynucleotide (b).

2. the polynucleotide of claim 1, the polypeptide shown in this polynucleotide encoding SEQ ID NO:2.

3. the polynucleotide of claim 1, wherein said polynucleotide are DNA.

4. the polynucleotide of claim 1, wherein said polynucleotide are RNA.

5. the polynucleotide of claim 1, wherein said polynucleotide are genomic dnas.

6. the polynucleotide of claim 1, these polynucleotide comprise 1 to 1209 Nucleotide shown in the SEQ ID NO:1.

7. the polynucleotide of claim 1, the soluble form of the polypeptide shown in its coding SEQ ID NO:2.

8. isolating polynucleotide, this polynucleotide comprise the member who is selected from as next group:

(a) a kind of polynucleotide, this polynucleotide encoding is a kind of by the mature polypeptide that is included in the dna encoding in No. 97128 preservation things of ATCC;

(b) a kind of polynucleotide, this polynucleotide encoding is by the DNA polypeptide expressed that is included in No. 97128 preservation things of ATCC;

(c) a kind of polynucleotide, this polynucleotide can with (a) or multi-nucleotide hybrid (b), and and they have at least 70% homogeny;

(d) polynucleotide passage of a kind of (a) and (b) or polynucleotide (c).

9. the polynucleotide of claim 8, wherein said polynucleotide encoding is by the DNA polypeptide expressed that is included in No. 97128 preservation things of ATCC.

10. the polynucleotide of claim 8, wherein said polynucleotide encoding is by the DNA polypeptide expressed that is included in No. 97128 preservation things of ATCC.

11. a carrier, this carrier comprises the DNA of claim 2.

12. one kind transforms with the carrier of claim 11 or the host cell of transfection.

13. a method of producing polypeptide, this method comprise the polypeptide of the host cell expression of Accessory Right requirement 12 by said dna encoding.

14. the method for the cell that a production can express polypeptide, this method comprises that the carrier with claim 11 transforms or the described cell of transfection.

15. a receptor polypeptides, this peptide species are selected from as next group:

(a) peptide species, this peptide species have deduced amino acid and its fragment, analogue and the derivative of SEQ ID NO:2; With

(b) a kind of cDNA encoded polypeptides and its fragment, analogue and derivative by No. 97128 preservation things of ATCC.

16. the polypeptide of claim 15, wherein said polypeptide has the deduced amino acid of SEQ ID NO:2.

17. the antibody of the polypeptide of a claim 15.

18. compound that activates the polypeptide of claim 15.

19. activated compound that suppresses the polypeptide of claim 15.

20. a therapeutic composition that is used to activate neuropeptide receptor comprises the compound of the claim 18 for the treatment of significant quantity.

21. a therapeutic composition that is used to suppress neuropeptide receptor comprises the compound of the claim 19 for the treatment of significant quantity.

22. the therapeutic composition of claim 20, wherein said compound is a peptide species.

23. the therapeutic composition of claim 21, wherein said compound is a peptide species.

24. one kind differentiate in conjunction with and activate the method for compound of the receptor polypeptides of claim 15, this method comprises:

Be provided at the recombinant host cell of the described receptor polypeptides of its surface expression, described acceptor is replied a kind of compound and is associated with second component of the bonded detectable signal of said receptor polypeptides with can providing;

Be enough to allow under compound and the said receptor polypeptides bonded condition multiple compound to be contacted with said host cell; With

By detect the signal that produces by said second component differentiate can with those compounds of receptors bind.

25. the agonist compound of differentiating by the method for claim 24.

26. a discriminating is in conjunction with the polypeptide of claim 15 and suppress the method for its activated compound, this method comprises:

Be provided at the recombinant host cell of the described receptor polypeptides of its surface expression, described acceptor is replied a kind of compound and is associated with second component of the bonded detectable signal of said receptor polypeptides with can providing;

Under the condition that is enough to allow in conjunction with said receptor polypeptides, the analytically detectable part of known bind receptor polypeptide is contacted with said host cell with multiple compound; With

Whether exist by detecting the signal that produces by part and polypeptide interaction, determine whether described part is attached on the described polypeptide.

27. the agonist compounds of differentiating by the method for claim 26.

28. whether a part of measuring on the polypeptide that can the unknown be attached to claim 15 can be connected to the method on the described polypeptide, this method comprises:

Under permission bonded condition, a kind of recombinant host cell at the described polypeptide of its surface expression is contacted with a kind of part to be identified, and detect the existence of the acceptor of any binding partner.

29. the method for claim 28 is wherein with before part to be identified contacts, from said cellular segregation receptor polypeptides or comprise the membrane component of this receptor.

30. a SCREENED COMPOUND is with the method for discriminating in conjunction with those compounds of the receptor polypeptides of claim 15, this method comprises:

Under the condition that allows to be attached on the acceptor, a kind of recombinant host cell at the described polypeptide of its surface expression is contacted with known analytically detectable part in conjunction with described acceptor with many candidate compounds; With

Discriminating can strengthen or suppress described part and be attached to those candidate compounds on the described acceptor.

31. the nucleic acid of the polypeptide of coding claim 15 is used for diagnosing purposes with the diagnostic reagent of not enough relevant disease of described polypeptide expression or disease susceptibility in preparation.

32. the polypeptide of claim 15, wherein said polypeptide are the soluble fragments of polypeptide, and part that can bind receptor.

33. the purposes of the polypeptide of claim 32 in a kind of diagnostic reagent of preparation.

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