CN1703625A - Drosophila g protein coupled receptors, nucleic acids, and methods related to the same - Google Patents

Abstract

The present invention provides Drosophila melanogaster GPCR (DmGPCR) polypeptides and polynucleotides which identify and encode such a DmGPCR. In addition, the invention provides expression vectors, host cells, and methods for its production. The invention also provides methods for the identification of homologues in other species and of DmGPCR agonists/antagonists useful as potential insecticides. The invention further provides methods for binding a DmGPCR, methods for identifying modulators of DmGPCR expression and activity, methods for controlling a population of insects with a DmGPCR antibody, a DmGPCR antisense polynucleotide, a DmGPCR binding partner or modulator, and methods of preventing or treating a disease or condition associated with an ectoparasite. Specifically, this invention discloses the matching of the orphan Drosophila short neuropeptide F receptor with its cognate peptide ligands.

Description

Fruit bat g protein coupled receptor, nucleic acid and correlation technique

Technical field

The present invention partly relates to the nucleic acid molecules of the novel fruit bat g protein coupled receptor of coding (DmGPCR), novel polypeptide, screening combines and/or regulates the test of DmGPCR activity with DmGPCR, method in conjunction with DmGPCR, reagent, for example DmGPCR antibody, detect the primer and the probe of encoding D mGPCR nucleotide sequence, the kit that comprises antibody of the present invention, primer and probe, comprise the composition of DmGPCR, DmGPCR binding partners and DmGPCR correctives and with the method for DmGPCR binding partners or correctives control insect colony.

Background technology

People and other life form are made up of living cells.The cell of life entity is linked up each other, and the mechanism of acquired information and stimulation is the cell-membrane receptor molecule of cell surface expression from environment.Identified many such acceptors, and determined their feature to become main receptor superfamily according to its structural motif with the signal transduction tagsort sometimes.These families include, but is not limited to part gated ion channels acceptor, depend on the ion channel acceptor of voltage, receptor tyrosine kinase, receptor protein tyrosine phosphatase and g protein coupled receptor.These acceptors are that extracellular signal is translated into the primary contact that cell physiological is replied.

G protein coupled receptor (being GPCR) has constituted a big superfamily of cell surface receptor, it is characterized in that an amino terminal extracellular domain, carboxyl terminal born of the same parents internal area and one stride the serpentine configuration of film 7 times.Therefore, these acceptors are also sometimes referred to as 7 and stride film (7TM) acceptor.These 7 membrane-spanning domains form in 3 born of the same parents' outer shrouds and 3 born of the same parents and encircle, and amino and carboxyl terminal domain.The outer part of the born of the same parents of acceptor has played identification and in conjunction with the effect of the outer binding partners (being part) of one or more born of the same parents, and the effect that part plays identification and communicates with each other with downstream effect thing molecule in the born of the same parents.

GPCR can combine with various parts, comprises calcium ion, hormone, chemotactic factor (CF), neuropeptide, neurotransmitter, nucleotide, lipid, odorant even photon.Natural, GPCR is important in normal (being unusual sometimes) function of many cell types.Generally see Strosberg, Eur.J.Biochem., 1991,196,1-10; Bohm etc., Biochem J., 1997,322,1-18.When its corresponding receptors bind of certain specific ligand, the common costimulatory receptor of this part and activate the different trimerization guanylic acid of a specificity in conjunction with regulating albumen (G albumen), in the born of the same parents of this albumen and acceptor partly or regional coupling.G albumen and then signal passed to intracellular effector molecule, thus stimulate or suppress the activity of this effector molecule.These effector molecules comprise adenosine cyclase of acid, phosphatidase and ion channel.The adenosine cyclase of acid is the enzyme relevant with the generation of second messenger molecule cAMP, InsP3 and diacylglycerol with phosphatidase.By these incidents that take place successively, the stimulation of the outer part of born of the same parents has produced in the born of the same parents by g protein coupled receptor and has changed.These acceptors all have characteristic primary structure, expression pattern, part binding pattern and the born of the same parents' internal effect system system of self separately.

Because the vital role of g protein coupled receptor in cell and the communication of its environment, these acceptors are attractive adjusting targets, for example can regulate by activation or these acceptors of antagonism.For the acceptor with known ligand, the evaluation of activator or antagonist can be especially at the effect that strengthens or suppress part.For example, some g protein coupled receptors in the disease genesis mechanism, work (for example some chemokine receptors as the collaborative acceptor of HIV may work in AIDS pathology takes place), therefore be in addition lack for the native ligand of this receptor take off, all be the attractive target that treatment is intervened.Other acceptor is owing to itself being to express in the tissue of the treatment attractive target of intervening or the cell type, also be the attractive target that treatment is intervened.The example of back one receptoroid comprises the acceptor that immunocyte is expressed, and they may be that inhibition autoimmune response or enhance immunity are replied the target of opposing pathogen or cancer; The acceptor of brain or other nervous organ and tissue expression may be treatment schizophrenia, depression, bipolar disorder or other neuropathic target.Back one receptoroid also can be used as to be identified and/or the sign of the cell subsets of the purifying cell sorting of fluorescent activation (for example by) expressed receptor.

Insect is considered to the primary pest in agricultural and the people family environment.Insect is also parasitic on one's body the animal and human, is referred to herein as epizoa, causes morbidity and dead.Insect is still to the carrier of plant, animal and human's class transmitted virus and parasitic disease.Therefore, for finding control insect colony, drive away and/or the new method of killing pathogenicity or parasite species has and continues and exigence.A kind of is by the use chemical agent by killing or making the method for insect paralysis control insect colony, is called pesticide, and they have the toxicity of selection to insect, also other invertabrate are had potential toxicity.Pesticide to agricultural product, comprises that the control of the insect of crops and livestock has great value to those at present.Pesticide also is used for the human residential environment, is used to control meadow and garden insect and the people is produced the insect of injury or puzzlement, for example stings the people or bites people's insect, fly and cockroach.Pesticide also has huge value for the disease that epizoa (comprise flea, louse, flat lice, tick and bite fly) in treatment or prevention domestic animal and the pet causes.Yet the chemical substance as pesticide is not best at present.Some have tangible toxicity to mammal, and the drug resistance to them also produces in some target insect simultaneously.Therefore, there are needs to novel selective insecticide with new role mechanism.

Known example with insect GPCR of neuropeptide part (see for example Li, etc., EMBO Journal, 1991,10,3221-3229; Li, etc., J.Biol.Chem., 1992,267,9-12; Monnier, etc., J.Biol.Chem., 1992,267,1298-1302; Vanden Broeck, etc., Int.Rev.Cytology, 1996,164,189-268; Guerrero, Peptides, 1997,18,1-5; Hauser, etc., J.Biol.Chem., 1997,272,1002-1010; Birgul etc., EMBO J., 1999,18,5892-5900; Torfs etc., J.Neurochem., 2000,74,2182-2189; With Hauser etc., Biochem.Biophys.Res.Comm., 1998,249,822-828; Larsen, etc., Biochem.Biophys.Res.Comm., 2001,286,895-901; Lenz, etc., Biochem.Biophys.Res.Comm., 2001,286,1117-1122; Kubiak etc., Biochem.Biophys.Res.Comm., 2002,291,313-320; Staubli etc., Proc.Natl.Acad.Sci.USA, 2002,99,3446-3451; Garczynski etc., Peptides, 2002,23,773-780), Holmes etc., Insect Molecular Biology, 2000, (5), 457-465; Proc.Natl.Acad.Sci.USA such as Cazzamali, 2002,99,12073-12078; Cazzamali etc., Biochem.Biophys.Res.Comm., 2002,298,31-36; Radford etc., J.Biol.Chem.2002,277,38810-38817; Park etc., Proc.Natl.Acad.Sci.USA, 2002,99,11423-11428; Kreienkamp etc., J.Biol.Chem, 10.1074/jbc.M206931200 (online delivering on August 6th, 2002) and Mertens, etc., Biochem.Biophys.Res.Comm., 2002,297,1140-1148.Nearest related application: Ebens, Allen James, Jr.; Torpey, Justin; Keegan, Kevin Patrick.Nucleic acids andpolypeptides of Drosophila melanogaster G protein-coupled receptor andtheir use as pesticidal and pharmaceutical targets.PCT Int.Appl. (2001), 43 pp.CODEN:PIXXD2 WO, 0170981 A2,20010927 CAN 135:268323 AN2001:713564 CAPLUS.Kravchik, Anibal.Drosophila G protein-coupledreceptors, genomic DNA and cDNA molecules encoding GPCR proteins, and theiruses as insecticidal targets.PCT Int.Appl. (2001), 392 pp.CODEN:PIXXD2 WO, 0170980 A2,20010927 CAN 135:269068 AN 2001:713563CAPLUS).

Usually in invertabrate (for example insect), find to be generally 4-12 amino acid whose one big class peptide be a class neuropeptide that is known as FMRF acid amides-related peptides (being FaRP).The name of prototype FMRF acid amides (FMRFa) peptide is owing to having " FMRF " consensus amino acid sequences at its C end, usually by (F, Y) (M, V, I, L) R (F, Y) NH2 formation.As neuropeptide, these molecules needing to participate in the life process of controlled neuromuscular activity.Though the effect of some neurotransmitters and neuregulin (comprising neuropeptide) that shown is the part of acceptor, will not identify FaRP neuropeptide so far as the part of GPCR.

The fruit bat peptide and mammal tachykinin (tachykinins) structurally associated that contain conservative FXGXR-acid amides motif, therefore a speech " fruit bat tachykinin (drotachykinin) " (Siviter etc., J.Biol.Chem., 2000 have been produced, 275 (30), 23273-23280).The fruit bat tachykinin has powerful effect of stimulation (subcutaneous) to the contraction of insect intestines.

Leucokinin is a component cloth insect hormone widely, and it stimulates enterocinesia and intestinal tube liquid secretion speed.In intestinal tube, their main effect be by with basolateral membrane on receptors bind improve cl permeability.Only raising cellular calcium effect in satellite cell of leucokinin (O ' Donnell etc., Am.J.Physiol., 1998,43, R1039-R1049).

Allatostatin is the insect neurohormone of one group of important various function of control, is included in the synthetic and not reproduction of insect of the same race of the juvenile hormone that plays central role in the metamorphosis.First kind of fruit bat allatostatin the earliest, Ser-Arg-Pro-Tyr-Ser-Phe-Gly-Leu-NH2 (being fruit bat inhibin (drostatin)-3) (SEQ IDNO:165) is (Birgul etc., EMBO J., 1999 of separating from the extract of fruit bat head, 18,5892-5900).Recently, cloned a kind of fruit bat allatostatin preprohormone gene, its four kinds of fruit bat allatostatin: Val-Glu-Arg-Tyr-Ala-Phe-Gly-Leu-NH2 (fruit bat inhibin-1) (SEQ IDNO:163) that encode, Leu-Pro-Val-Tyr-Asn-Phe-Gly-Leu-NH2 (fruit bat inhibin-2) (SEQ ID NO:164), Ser-Arg-Pro-Tyr-Ser-Phe-Gly-Leu-NH2 (fruit bat inhibin-3) (SEQ ID NO:165), and Thr-Thr-Arg-Pro-Gln-Pro-Phe-Asn-Phe-Gly-Leu-NH2 (fruit bat inhibin-4) (SEQ ID NO:166) (Lenz etc., Biochem.Biophys.Res.Comm., 2000,273,1126-1131).First kind of fruit bat allatostatin acceptor be by clones such as Birgul, show by fruit bat inhibin-3 by its function of Gi/Go pathway activation (Birgul etc., EMBO J.1999,18,5892-5900).Cloned recently second kind of supposition fruit bat allatostatin acceptor (being DARII) (Lenz etc., Biochem.Biophys.Res.Comm., 2000,273,571-577).DARII receptor cdna (accession number AF253526) coding and first kind of closely-related albumen of fruit bat allatostatin acceptor.Recently, we (Larsen, etc., Biochem.Biophys.Res.Comm., 2001,286,895-901) and other people (Lenz, etc., Biochem.Biophys.Res.Comm., 2001,286,1117-1122) disclosed the functional activation of allatostatin to DARII.Recently, cloned fruit bat allatostatin C type preprohormone, its coding fruit bat allatostatin C:Gln-Val-Arg-Tyr-Gln-Cys-Tyr-Phe-Asn-Pro-Ile-Ser-Cys-Ph e-OH (Williamson etc., Biochem.Biophys.Res.Comm., 2001,282,124-130).Mature peptide should have pGlu at the N-end, and this is the result of the terminal Gln cyclisation of N-, obtains: pGlu-Val-Arg-Tyr-Gln-Cys-Tyr-Phe-Asn-Pro-Ile-Ser-Cys-Phe-OH (SEQ ID NO:183); And the disulfide bond between Cys6 and Cys13, similar with hawkmoth (Manduca sexta) C type allatostatin pGlu-Val-Arg-Phe-Gln-Cys-Tyr-Phe-Asn-Pro-Ile-Ser-Cys-Phe-OH (SEQ ID NO:182), they are only at 4 different (Phe4 is to Try4) (Kramer etc., Proc.Natl.Acad.Sci.USA, 1991,88,9458-9462).Strong and the lasting contraction of muscle that Nichols etc. have disclosed fruit bat allatostatin-C suppresses, be called platform line (FLT) peptide (Peptides such as Nichols, 2002,23,787-794).As far as our knowledge goes, do not identify the acceptor of any insect C type allatostatin so far.

Sulfanilamide (SN) kassinin kinin (sulfakinin) is a class insect Tyr-sulfonation neuropeptide.The similarity of (muscle effect, the enzyme that stimulates digestion discharges) on their demonstrations and vertebrate peptide gastrin and cholecystokinin sequence and the function.In fruit bat, identify coding two kinds of sulfanilamide (SN) kassinin kinins (being also referred to as fruit bat sulfanilamide (SN) kassinin kinin) DSKI[Phe-Asp-Asp-Tyr (SO3H)-Gly-His-Met-Arg-Phe-acid amides] (SEQ ID NO:160) and DSKII[Gly-Gly-Asp-Asp-Gln-Phe-Asp-Asp-Tyr (SO3H)-Gly-His-Met-Arg-Phe-acid amides] gene (Nichols of (SEQID NO:161), Mol.Cell Neuroscience, 1992,3,342-347; Nichols etc., J.Biol.Chem., 1988,263,12167-12170).In all sulfanilamide (SN) kassinin kinins that identify in the caste that terminal seven peptide sequence Asp-Tyr (the SO3H)-Gly-His-Met-Arg-Phe-acid amides of C-(SEQ ID NO:162) extensively separate evolving so far is identical.The conservative property of this seven peptide sequence comprises the existence of sulfonation Tyr residue in extensively various classification of insect group, infers " activity core " importance (the Nachman ﹠amp on function that has reflected this close flesh; Holman, in Insect Neuropeptides:Chemistry, Biology and Action, Menn, Kelly ﹠amp; Massler compiles, American Chemical Society, and Washington, D.C., 1991, pp.194-214).Recently, the orphan receptor (DmGPCR9) that we identify fruit bat is a fruit bat sulfanilamide (SN) kinin receptor (being called DSK-R1), and with the activating peptide of itself and fruit bat, the fruit bat sulfanilamide (SN) kassinin kinin-1 that Met5 → Leu modifies, Asp-Tyr (SO3H)-Gly-His-Leu-Arg-Phe-acid amides (SEQ ID NO:157) mates (Kubiak etc., Biochem.Biophys.Res.Comm., 2002,291,313-320).New non-orphan fruit bat GPCR comprises PRX amidated peptide, CCAP, corazonin and AKH (Park etc., Proc.Natl.Acad.Sci.USA, 2002,99,11423-11428; Cazzamali etc., Biochem.Biophys.Res.Comm., 2002,298,31-36); Leucokinin (Radford etc., J.Biol.Chem.2002,277,38810-38817); Fruit bat inhibin-C (Kreienkamp etc., J.Biol.Chem, 10.1074/jbc.M206931200 (online publication on August 6 in 2002)); The FMRF acid amides (Proc.Natl.Acad.Sci.USA such as Cazzamali, 2002,99,12073-12078); With neuropeptide F (Mertens, etc., Biochem.Biophys.Res.Comm., 2002,297, acceptor 1140-1148).

Summary of the invention

The present invention relates to novel polypeptide in the fruit bat, the surprising discovery of this paper called after DmGPCR (fruit bat g protein coupled receptor), it shows other neuropeptide GPCR homology in various degree.The invention provides these genes of unknown G-G-protein linked receptor before of encoding, the DmGPCR polypeptide of these gene codes; The antibody of this polypeptide; Use the kit of polynucleotide and polypeptide, and the method for preparing and use previous described above-mentioned substance.DmGPCR may play the effect of key component, for example regulates neuropeptide combination and/or signal transmission.Therefore, DmGPCR can be used for searching can change and/or control combination, and/or passes through the novel formulation of neuropeptide or other material Transfer signal.These and other aspect of invention describes in detail hereinafter.

In certain embodiments, the invention provides purifying and the DmGPCR polypeptide that separates, it has the IDNOs:2 at SEQ, and 4,6,8,10,12,14,16,18,20,22, or one of 24 amino acid sequences of listing or its contain the fragment of DmGPCR specificity epitope." epitope specificity " refers to that the part of DmGPCR acceptor can be discerned by the DmGPCR specific antibody, as detailed below.One embodiment of the present of invention comprise purifying and the polypeptide that separates, and have SEQ ID NOs:2,4,6,8,10,12,14,16,18,20,22, or the complete amino acid sequence of listing in 24, shown in hereinafter table 4.These amino acid sequences are to infer and come from the polynucleotide sequence of encoding D mGPCR (SEQ ID NOs:1,3,5,7,9,11,13,15,17,19,21, or 23, table 4 sees below).The singulative of term used herein " DmGPCR " be meant comprise list below by each of 10 seed amino acid sequences of each polynucleotide sequence coding.

Though the sequence that provides is concrete fruit bat sequence, the present invention also will comprise allele variant, vertebrate and the invertabrate form of DmGPCR in its scope.

In certain embodiments, the invention provides purified polynucleotides (no matter for example cDNA, genomic DNA, synthetic property DNA, RNA or its combination is strand or two strands), nucleotide sequence with code book invention amino acid sequence of polypeptide.These polynucleotide are used for recombinant expressed acceptor, also are used for detecting cell receptor expression (promptly with Northern hybridization and in situ hybridization test).These polynucleotide also are used to design antisense and other molecule, to suppress or the expression of regulation and control DmGPCR in cultured cell, tissue or animal.Special eliminating is complete separation, the nonrecombinant natural dyeing body of host cell from polynucleotide definition of the present invention.Polynucleotide of the present invention can have SEQ ID NOs:1, and 3,5,7,9,11,13,15,17,19,21, or arbitrary sequence of listing in 23.Should understand because the degeneracy of general genetic code exists many other polynucleotide sequences also can encode and has SEQ ID NOs:2,4,6,8,10,12,14,16,18,20,22, or 24 arbitrary DmGPCR that list sequence.

The present invention also provides the purifying of the polynucleotide sequence that contains the encoding mammalian polypeptide and the polynucleotide that separate, and wherein polynucleotide are under following hybridization conditions:

A) in the hybridization solution that contains 50% formamide, 1%SDS, 1M NaCl, 10% sulfuric acid glucose 42 ℃ hybridization 16 hours; With

B) containing 0.1%SSC, 60 ℃ of rinsings are 2 times in the washing lotion of 1%SDS, and each 30 minutes,

And have SEQ ID NOs:1,3,5,7,9,11,13,15,17,19,21, or the multi-nucleotide hybrid of 23 arbitrary sequences of listing.

Hybridization conditions should be in the presence of other nucleic acid molecule, only with the condition of gene recombination.Under stringent hybridization condition, only highly complementary nucleic acid array hybridizing.Such hybridization conditions can prevent to have in 20 continuous nucleotides the nucleic acid hybridization of 1 or 2 mispairing.

In certain embodiments, the invention provides the carrier that contains polynucleotide of the present invention.These carriers for example are used at the host cell polynucleotide that increase, to produce the polynucleotide of useful quantity.In certain embodiments, carrier is an expression vector, and polynucleotide of the present invention are connected with the polynucleotide navigability that contains expression control sequenc in this carrier.These carriers can be used for reorganization and produce polypeptide of the present invention.

In certain embodiments, the invention provides with polynucleotide of the present invention or carrier of the present invention and transform or the host cell of transfection (stable or instantaneous).As mentioned above, these host cells can be used for the polynucleotide that increase, and also can be used for expressing DmGPCR polypeptide or its fragment of this polynucleotide encoding.

In another embodiment, the invention provides the method that produces DmGPCR polypeptide (or its fragment), comprise step: in nutrient medium, cultivate host cell of the present invention, isolated polypeptide or its variant from cell or nutrient culture media.Because DmGPCR is 7 transmembrane receptors, should understand in some application, for example in some activity test, separation may relate to and separate the cell membrane that contains this polypeptide, and other may need to separate more completely in using.

Should understand the outer epi-position of born of the same parents for producing and screening antibody and other energy and acceptor, for example the binding compounds of DmGPCR combination is useful especially.Therefore in another embodiment, the invention provides purifying and the polypeptide that separates, contain at least one extracellular domain (that is, one of the terminal extracellular domain of N-or 3 born of the same parents' outer shrouds) of DmGPCR, for example the terminal extracellular domain of the N-of DmGPCR.The present invention also comprises the polypeptide of purifying, contains the membrane-spanning domain of DmGCPR, the born of the same parents' outer shroud that is connected with membrane-spanning domain of DmGPCR, ring in the born of the same parents that DmGPCR is connected with membrane-spanning domain, the terminal cytoplasmic region of the C-of DmGPCR, and fusion.These fragments can be the continuous parts of natural receptor.Yet, will also be understood that the knowledge of DmGPCR gene provided herein and protein sequence allows discontinuous each zone reorganization in the native protein.

In another embodiment, the invention provides specific antibody to DmGPCR of the present invention.Antibody specificity as detailed below.Yet what particularly point out is that the described polypeptide of previous document produces, and can be regarded as " cross reactivity " antibody with the antibody of the lucky cross reaction of DmGPCR (for example because all lucky in two peptide species have a similar epi-position).These cross reacting antibodies are not DmGPCR's " specificity " antibody.Determine that a kind of antibody is whether specific or can not play cross reaction with another kind of known receptor to DmGPCR, arbitrary the carrying out in available several tests, Western blotting test for example, this is well known in the art.In order to identify the cell of expressing DmGPCR and to regulate the DmGPCR-ligand-binding activity, can use the antibody of energy specificity in conjunction with the outer epi-position of born of the same parents of DmGPCR.

In a kind of variation, the invention provides monoclonal antibody.The hybridoma that produces these antibody also is regarded as an aspect of of the present present invention content.

In another kind changes, the invention provides the cell-free composite that contains polyclonal antibody, wherein at least a antibody is to the specific antibody of the present invention of DmGPC.The antiserum that separates from animal is an exemplary composition, is a kind of composition that contains sero-fast antibody component, and this component is suspended in water or other thinning agent, excipient or the carrier.

In another related embodiment, the invention provides specific anti-idiotype to specificity DmGPCR antibody.

As everyone knows, antibody contains the less antigen binding domain of can enough chemical methodes or separating by recombinant technique.These functional domains itself are useful DmGPCR binding molecules, also can merge with toxin or other polypeptide.Therefore, in another embodiment, the invention provides the fragment that contains the DmGPCR specific antibody, wherein fragment and polypeptide combine with DmGPCR.By non-limiting example, polypeptide provided by the invention is single-chain antibody, CDR-grafted antibody and humanized antibody.

Also comprise composition in the scope of the invention, contain with materia medica for example on polypeptide of the present invention, polynucleotide or the antibody of acceptable carrier allotment.

The present invention also provides the method for using antibody of the present invention.For example, the invention provides the method for regulating the combination of DmGPCR part, comprise step: under the condition of antibody and receptors bind, make DmGPCR and the narrow spectrum antibody of DmGPCR is contacted.

The invention provides and in individuality, induce at having the SEQ of being selected from ID NO:2,4,6,8,10,12,14,16,18, the method for the immune response of 20,22 and 24 polypeptide of sequence.This method comprises the polypeptide of individuality being used enough induce immune response amounts.

The present invention also provides the test of identifying the compound that combines with DmGPCR.A kind of test comprises step: the composition that contains DmGPCR can be contacted in conjunction with the compound of DmGPCR with suspection; (b) measure combining between this compound and DmGPCR.In a kind of variation, said composition contains the cell of surface expression DmGPCR.In another kind changes, use the DmGPCR that separates or contain the cell membrane of DmGPCR.This combination can directly be measured, and the compound by usage flag for example, or can pass through several technical measurements indirectly comprises the intracellular signal transmission (or measuring variation that the DmGPCR signal transmits level) of measuring the DmGPCR that this compound induces.

The present invention also provides the method that DmGPCR is combined with binding partners.The method comprising the steps of: the composition that contains DmGPCR is contacted with binding partners; (b) binding partners is combined with DmGPCR.For example, DmGPCR can be DmGPCR1 (SEQ ID NO:1), DmGPCR5 (SEQ ID NO:9), DmGPCR7 (SEQ ID NO:17), or DmGPCR8 (SEQ ID NO:19).Binding partners can be for example fruit bat tachykinin, leucokinin or allatostatin-C.Fruit bat tachykinin (DTK) can be DTK-1 (SEQ IDNO:169) for example, Met8-DTK-2 (SEQ ID NO:170), DTK-2 (SEQ ID NO:171), DTK-3 (SEQID NO:172), DTK-4 (SEQ ID NO:173), and DTK-5 (SEQ ID NO:174).Leucokinin (LK) can be LK-I (SEQ ID NO:175) for example, LK-V (SEQ ID NO:176), LK-VI (SEQ IDNO:177), and LK-VIII (SEQ ID NO:178), culex kassinin kinin (Culekinin) (SEQ ID NO:179), mollusc leucokinin sample peptide, snail kassinin kinin (lymnokinin) is (SEQ ID NO:180) (PSFHSWSa), with fruit bat leucokinin sample peptide DLK-1 (NSVVLGKKQRFHSWGa) (SEQ ID NO:181), DLK-2 (pGlu-RFHSWGa) (SEQ ID NO:182) and DLK-2A (QRFHSWGa) (SEQ ID NO:183).Allatostatin (AST) can be for example AST-C (SEQ ID NO:184) or DST-C (SEQ IDNO:185).Other binding partners includes but not limited to SEQ ID NO:186 and SEQ ID NO:187.

The present invention also comprises the correctives of identifying combination between DmGPCR and the DmGPCR binding partners, comprises step: the DmGPCR binding partners is contacted when having and not existing the correctives compound of supposition with the composition that contains DmGPCR; (b) combination between detection binding partners and the DmGPCR; (c) according to the combination in the presence of the correctives of certain supposition between binding partners and the DmGPCR, compare minimizing or increase with the combination under the correctives that does not have this supposition, identify the adjusting compound or the correctives compound of this supposition.For example, DmGPCR can be DmGPCR5 (SEQ ID NO:9), DmGPCR7 (SEQ ID NO:17), or DmGPCR8 (SEQ ID NO:19).Binding partners can be for example fruit bat tachykinin, leucokinin or allatostatin.Fruit bat tachykinin (DTK) can be DTK-1 (SEQ ID NO:169) for example, Met8-DTK-2 (SEQ ID NO:170), DTK-2 (SEQ ID NO:171), DTK-3 (SEQ ID NO:172), DTK-4 (SEQ ID NO:173), and DTK-5 (SEQ ID NO:174).Leucokinin (LK) can be LK-I (SEQ ID NO:175) for example, LK-V (SEQ ID NO:176), LK-VI (SEQ ID NO:177), and LK-VIII (SEQ ID NO:178), culex kassinin kinin (SEQ ID NO:179), mollusc leucokinin sample peptide snail kassinin kinin (PSFHSWSa) (SEQ ID NO:180), with fruit bat leucokinin sample peptide DLK-1 (NSVVLGKKQRFHSWGa) (SEQ ID NO:181, DLK-2 (pGlu-RFHSWGa) (SEQ IDNO:182), and DLK-2A (QRFHSWGa) (SEQ ID NO:183).Allatostatin (AST) can be AST-C (SEQ ID NO:184) for example, or DST-C (SEQ ID NO:185).In a kind of variation, said composition contains the cell at its surface expression DmGPCR.In another kind changes, use DmGPCR or contain the cell membrane of DmGPCR.In conjunction with can directly measuring, the compound by usage flag for example, or can be indirectly by several technology comprise the intracellular signal transmission (or measuring variation that the DmGPCR signal transmits level) of measuring the DmGPCR that this compound induces.For example, [35S] GTP γ S of available agonist induction measures this function in conjunction with test, cAMP test (inducing of producing of cAMP or suppress) or with fluorescence imaging flat board reading machine (FLIPR) assay determination cellular calcium level.

Can stimulate the DmGPCR binding partners of DmGPCR activity to can be used as activator, strengthen or prolong the transmission of DmGPCR signal, thereby disturb normal activated receptors signal pipeline.The DmGPCR binding partners that seals ligand-mediated DmGPCR signal transmission can be used as antagonist, interferes the transmission of normal DmGPCR signal and damages receptor-mediated effect.In addition, DmGPCR correctives and DmGPCR polynucleotide and polypeptide can be used for using in the diagnostic test of the disease of DmGPCR increased activity or damage and situation.

In yet another aspect, the invention provides the treatment disease that causes of epizoa or the method for unusual condition, needs the individual physical efficiency adjusting of this treatment to be selected from SEQ ID NO:2,4,6,8,10,12,14,16,18,20,22, or the material of 24 epizoa polypeptide active.

Situation or the useful material of disease treatment that epizoa is caused can show positive findings in measuring one or more in vitro tests for the treatment of described disease or unusual corresponding activity.The material that can regulate this polypeptide active includes but not limited to antisense oligonucleotides, activator and antagonist and antibody.

On the other hand, the invention provides a peptide species in the test sample, cause the method for disease or unusual diagnostic tool as epizoa, wherein the method comprising the steps of: sample is contacted with nucleic acid probe, and this probe is selected from SEQ ID NO:2,4 with coding under the cross experiment condition, 6,8,10,12,14,16,18,20,22, or the target set nucleic acid area hybridization of 24 polypeptide, described probe contains the nucleotide sequence of coded polypeptide, its fragment and/or the complementary series of sequence and fragment; (b) detector probe: the existence of target area hybridization or quantity are as the situation index.

The specimen that is fit to nuclei acid probe method of the present invention comprises the nucleic acid extractive of cell for example or cell, or biological fluids.The sample that is used for said method can change according to test form, detection method and the tissue that will test, cell or extract character.The method of the nucleic acid extractive of preparation cell is known in the art, and can change easily, with the sample that obtains to adapt with used method.

The invention provides the homologue of DmGPCR in certain embodiments, for example the mammal homologue.The mammal homologue of DmGPCR can be expressed in tissue, and these tissues include but not limited to neural system tissue, pancreas (particularly islet tissue), hypophysis, skeletal muscle, adipose tissue, liver, intestines and stomach and thyroid gland.

In certain embodiments, the invention provides the method for identifying DmGPCR mammal homologue, comprise step: with being selected from SEQ ID NOs:1,3,5,7,9,11,13,15,17,19,21, screen mammalian nucleic acid database or nucleic acid library with 23 nucleic acid or its part, and determine whether the part and this sequence homology in database or library.

Another aspect of the present invention provides by insect being used the binding partners or the correctives of DmGPCR polynucleotide or polypeptide, and DmGPCR expresses or activity to change, thus the method for control insect colony.For example, insect can be selected from fly, fruit bat, tick, flea, louse, flat lice and bite fly.

The DmGPCR binding partners can be the fruit bat tachykinin (for example, DTK-1 (SEQ ID NO:169), Met8-DTK-2 (SEQ ID NO:170), DTK-2 (SEQ ID NO:171), DTK-3 (SEQ ID NO:172), DTK-4 (SEQ ID NO:173), and DTK-5 (SEQ ID NO:174)), leucokinin (e.g., LK-I (SEQ ID NO:175), LK-V (SEQ ID NO:176), LK-VI (SEQ ID NO:177), and LK-VIII (SEQ ID NO:178), culex kassinin kinin (SEQ ID NO:179), mollusc leucokinin sample peptide snail kassinin kinin (PSFHSWSa) (SEQ ID NO:180), DLK-1 (SEQ ID NO:181), DLK-2 (SEQ ID NO:182), and DLK-2A (QRFHSWGa) (SEQ ID NO:183)), or allatostatin (AST-C (SEQ ID NO:184 or DST-C SEQ ID NO:185)).Other binding partners includes but not limited to SEQ ID NO:186 and SEQ ID NO:187.The DmGPCR correctives can be anti-DmGPCR antibody or DmGPCR antisense polynucleotides.

An alternative embodiment of the invention provides the disease that prevention or treatment epizoa cause or the method for the patient's condition in the host, comprise binding partners or the correctives of individuality being used DmGPCR polynucleotide or polypeptide, to regulate expression or the activity of DmGPCR.

Those skilled in the art can comprise and understand further feature of the present invention or variation in the detailed description that these features all are parts of the present invention from the entire chapter application.Similarly, the feature of invention as herein described can be reassembled into other embodiment, and they also are parts of the present invention, no matter whether these combination of features are above as part of the present invention or the referred mistake of embodiment.In addition, have only those restrictions to key of the present invention described herein just should be considered to crucial; Variation of the present invention is not as long as have to be crucial, just to be considered to be a part of the present invention in this article referred.

Preferred implementation describes in detail

The present invention provides the separation and the purified polynucleotides of coding fruit bat g protein coupled receptor (DmGPCR) or its part especially, the carrier that contains these polynucleotide, by these carrier transformed host cells, the method for preparing DmGPCR, use above-mentioned polynucleotide and carrier, separate and the method for the DmGPCR of purifying, the method of the method for the compound of screening adjusting DmGPCR activity and mammal, vertebrate or the invertabrate DmGPCR homologue of evaluation DmGPCR.

Various definition have been carried out in the presents.Most of literal has those skilled in the art and gives their implication.Special herein below or the literal that limits elsewhere the implication that provides under the situation as integral body of the present invention is provided, normally those skilled in the art are intelligible.

Should understand when listing sequence set, its combination or subgroup are also considered especially.For example, the present invention includes combination or subgroup, include but not limited to SEQ ID NO:1 and 3 for describing " SEQ ID NOs:1,3,5,7,9,11,13,15,17,19,21, or 23 ", should understanding; 1 and 5; 1,3 and 5 etc.

Term " synthetic " used herein and that this area is understood refers to what pure chemical method produced, with respect to the polynucleotide of enzyme process generation.Therefore " fully " synthetic dna sequence dna is to produce by chemical method fully, and " part " synthetic DNA comprises that those DNA that obtain only partly produce with chemical method.

Term " district " refers to a physically continuous part of biomolecule primary structure.For albumen, the zone refers to the continuous part of this protein amino acid sequence.

Term used herein " functional domain (domain) " refers in the biomolecule the known of this biomolecule or the contributive structure division of function suspected.Functional domain can extend jointly with its zone or part.Functional domain also can mix and the specific region, or the part of all or part of different biomolecule that should the zone.The example of gpcr protein functional domain includes but not limited to born of the same parents' outer (N-end), strides film and kytoplasm (being the C-end) functional domain, and they and the regional of the similar name of GPCR extend jointly; In GPCR7 TMD each; With each the ring section that is connected adjacent TMD (the outer and interior ring of born of the same parents of born of the same parents).

Direct or indirect combination is indicated or disclosed to term used herein " activity "; The influence reaction, but the various measurement indexes that promptly reaction of some contact or stimulation had measurable effect, for example comprise, the affinity that compound and polypeptide of the present invention or polynucleotide directly combine, or for example after some stimulation or incident to the mensuration of the amount of upstream or downstream egg white matter or other function.

Term used herein " antibody " means complete antibody and Fab, Fab ', F (ab ') ₂, Fv and other fragment.Complete antibody comprises monoclonal antibody, mouse monoclonal antibody for example, chimeric antibody, people's antibody and humanized antibody.

Term used herein " combination " refers to physics or the chemical interaction between two kinds of protein or compound or associated protein or compound or its combination.In conjunction with comprising ion, nonionic, hydrogen bond, Van der Waals force, hydrophobic effect etc.Physics interact (combination) can be direct or indirect, be indirectly by or the combination owing to the effect of another kind of protein or compound.Directly in conjunction with mean not by or be not because the effect of another kind of protein or compound, the interaction by other concrete chemical intermediate.

Term used herein " compound " refers to any appraisable chemical substance or molecule, includes but not limited to micromolecule, peptide, protein, sugar, nucleotide or nucleic acid, and these compounds can be natural or synthetic.

Term used herein " complementation " refers to the Hua Sheng-Ke Like base pairing between the nucleotide units of a nucleic acid molecules.

Term used herein " contact " means makes the direct or indirect physics of compound combine near polypeptide of the present invention or polynucleotide.Described polypeptide or polynucleotide can be formulated in damping fluid, salt, solution of any amount etc.Contact for example comprises, this compound is placed contain coding GPCR or the nucleic acid molecules of its fragment or beaker, microtiter plate, Tissue Culture Flask or the microarray of polypeptide, for example in the genetic chip etc.

Phrase used herein " homologous nucleotide sequence " or " homologous amino acid sequence " or its variant refer to have the sequence of the homology feature of specified percentage at least at nucleotide level or amino acid levels.The homology nucleotide sequence comprises the sequence of those coded protein isotypes.Because for example alternately montage of RNA, these isotypes can be expressed in unified biological different tissues.In addition, isotype can be encoded by different genes.The homology nucleotide sequence comprises the nucleotide sequence of the protein of the species (include but not limited to mammal) of coding except insect.The homology nucleotide sequence also includes but not limited to the mutation-ure of the nucleotide sequence that naturally occurring allele variant and this paper are listed.Yet the homology nucleotide sequence does not comprise the nucleotide sequence of other known GPCR that encodes.The homology amino acid sequence comprises the amino acid sequence that those coding conserved amino acids replace and has the neuropeptide combination and/or the active polypeptide of signal transmission.Yet homologous amino acid sequence does not comprise the amino acid sequence of other known GPCR that encodes.Can by breach program for example (Wisconsin sequence analysis software bag, Unix the 8th edition, GeneticsComputer Group, University Research Park, Madison WI), uses default setting to determine homology percentage.This program use Smith and Waterman algorithm (Adv.Appl.Math., 1981,2,482-489 is in this complete introducing for your guidance).

Term used herein " separation " nucleic acid molecules refers to the nucleic acid molecules (DNA or RNA) that is removed from its natural surroundings.The example of isolated nucleic acid molecule includes but not limited to the recombinant DNA molecules that comprises in the carrier, the recombinant DNA molecules in the heterologous host cell for this reason, nucleic acid molecules and the synthetic property DNA or the RNA molecule of part or basic purifying.

Term used herein " adjusting " means the raising or the minimizing of amount, quality or the effect of given activity or protein.

Term used herein " activity of enhancing " refers to the activity that improves.Term " impaired activity " refers to the activity that descends.

Term used herein " oligonucleotides " refers to the nucleotide residue of a series of connections, and their base quantity enough is used to PCR (PCR).This weak point sequence is based on (or according to down person's design) genome or cDNA sequence, is used for increasing, verify or discloses identical, similar or complementary DNA or the RNA existence at specific cells or tissue.Oligonucleotides comprises having at least about 10 nucleotide, about 50 nucleotide of as many as, the dna sequence dna of preferably about 15-30 nucleotide.They be chemosynthesis and can be used as probe.

Term used herein " probe " but refer to the nucleotide sequence of variation length, preferably, decide on purposes at least about 10 to 6,000 nucleotide at the most.They are used to detect identical, similar or the complementary nucleic acid sequence.Usually obtain the probe of length from natural or recombinant sources, have high degree of specificity, than oligomer hybridization slowly many.They can be strand or two strands, and have selectivity through well-designed at PCR, based on hybond membrane or ELISA sample technology.

When referring to polynucleotide, " part " or " fragment " comprises having at least 14,16,18,20,25,50 of fragment of deriving or reference polynucleotide partly, or the polynucleotide sequence of 75 continuous nucleotides.When referring to polypeptide, " part " or " fragment " comprises having at least 5,10,15,20,25,30,35 of fragment of deriving or reference polypeptide partly, or the peptide sequence of 40 continuous amino acids.

The possibility that term " prevention " refers to reduce biological contact or unusual condition takes place.

Term " control insect colony " or its variant refer to increase or reduce the quantity of insect in the colony.For example, the method for control insect colony comprises the quantity that increases useful insect in the given insect colony and reduces harmful insect quantity in the given insect colony.

Term " treatment " refers to have curative effect or alleviates or stop unusual in the biosome to small part.

Term used herein " individuality " refers to insect, vertebrate, invertabrate and mammal.

Term " curative effect " refers to suppress or activate the factor that causes or be responsible for unusual or normal condition.Curative effect has been alleviated one or more symptoms unusual or normal condition to a certain extent.Curative effect can refer to following one or more: the increase of (a) cell proliferation, growth and/or differentiation; (b) inhibition of cell death (promptly slow down or stop); (c) inhibition of Tui Huaing; (d) one or more symptoms relevant have been alleviated in a way with situation; (e) function of the influenced cell mass of enhancing.Can evaluation as described herein show at unusual or normal condition compounds effective.

The situation of the biology for the treatment of can be unusual or normal.Term " unusual condition " refers to that the function of the cell or tissue that some is biological run counter to the normal function in this biosome.For example, unusual condition can be relevant with cell proliferation, cell differentiation, cell signal transmission or cell survival.

Phrase " normal condition " refers to the normal function of biological cell or tissue.For example, normal condition can be relevant with cell proliferation, cell differentiation, cell signal transmission or cell survival.

Term administering " refer to a kind of method of compound being mixed biological cell or tissue.When in vivo or when having this biological cell or tissue outside the biosome, can prevent, treat or induce its state.Can outside biosome, maintain in the Tissue Culture Dish cell or cultivation.For the cell in the biosome, there is the method much be used for administered compound in this area, include but not limited to that oral cavity, peritonaeum are interior, subcutaneous, injection and gasoloid administration.For the outer cell of biosome, there is the method for many administered compounds in this area, includes but not limited to cell microinjection technology, transformation technology and carrier technology.

Also can prevent, treat or induce situation by the cell administered compound that must change the bion signal transduction pathway to one group.Can monitor the effect of administered compound then to biological function.Individuality can be for example mammal, for example mouse, rat, rabbit, cavy; Companion animals (for example dog or cat), animals (for example chicken, pig or ox), goat, horse, monkey, ape or people; Worm; Or insect.

" amplification " refers to compare the increase of DNA or RNA quantity in certain cell with normal cell." amplification " is that cell RNA can detect existence when being used for RNA, because there is not the baseline of RNA to express in some normal cells.In other normal cell, there is the expression of baseline values, therefore amplification is to detect with baseline values to compare 1-2 times at least in these cases, and is preferably more.

Term used herein " stringent hybridization condition " or " oxygen condition " refer in probe, primer or oligonucleotides and the hybridization of its target sequence, but the condition of other sequence hybridization of getting along well.Stringent condition depends on sequence, may be different under different situations.Long sequence is selectivity hybridization under higher temperature.Usually, the pyrolysis chain temperature (Tm) that is listed under specific ion intensity and the pH of stringent condition bit sequencing is low 5 ℃.Tm is that (under specific ion intensity, pH and nucleic acid concentration) has 50% to hybridize the temperature of balance with the probe of target sequence complementation and target sequence.Because normally excessive existence of target sequence, at Tm, 50% probe will be a balance.Usually, stringent condition is that salinity is for being less than the 1.0M sodion, about usually 0.01-1.0M sodion (or other salt), pH7.0-8.3, temperature for short probe, primer or oligonucleotides (for example 10-50 nucleotide) at least about 30 ℃, at least 60 ℃ of long probe, primer or oligonucleotides.Stringent condition also can be by adding destabilizing agent, and for example formamide is realized.

Amino acid sequence is with from left to right, and amino direction to carboxyl exists.Amino and carboxyl is not present in the sequence.Nucleotide sequence only with strand from 5 ' from left to right exist to 3 ' direction.Nucleotide and amino acid are the forms of recommending with the IUPAC-IUB biochemical nomenclature commission, or (for amino acid) exists with the trigram coding.

Polynucleotide

Genomic DNA of the present invention contains the protein coding region of polypeptide of the present invention, also comprises its allele variant.Should be understood that extensively that for many genes, genome is transcribed into the rna transcription thing, this transcript is through the one or many montage, and wherein the introne of transcript (being non-coding region) is removed, or " by cutting off ".In the art, can be by optional machine-processed montage, thus can remove the different RNA sequence, but still the rna transcription thing of encoding D mGPCR polypeptide is called " splice variant ", it also belongs to the present invention.The splice variant that the present invention understands is therefore by same original gene group dna sequence encoding, but from different mRNA transcripts.Allele variant is the modified forms of wild type gene sequence, and the condition that produces gene mutation in chromosome separation or contact causes this modification.Allele variant is the same with wild type gene, also is naturally occurring sequence (variant that exists with the non-natural of external manipulation generation is opposite).

The present invention also comprises the cDNA (second chain that carries out complementary strand usually subsequently is synthetic, obtains double-stranded DNA) that the RNA polynucleotide by reverse transcription encoding D mGPCR obtain.

The dna sequence dna of encoding D mGPCR is listed in SEQ ID NOs:1, and 3,5,7,9,11,13,15,17,19,21, or 23.DNA of the present invention can comprise duplex molecule and complementary molecule (" noncoding strand " or " complementary strand "), has the sequence that can not differentiate according to DNA basepairing rule and the coding strand of Hua Sheng-Ke Like.The present invention also comprises other polynucleotide of other any concrete DmGPCR polypeptide of the present invention of encoding, and the concrete polynucleotide sequence of their sequence and this paper is because well-known general purpose core gene-code degeneracy is different.

The present invention also comprises the homologue of the species such as mammal of DmGPCR DNA.The species homologue is known as " directly to homologue " sometimes, has at least 35%, at least 40% with DNA of the present invention usually, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homology.Usually, can be calculated to be at collating sequence and (as needs) with respect to " homology " percentage of polynucleotide of the present invention and to introduce breach, the percentage of nucleotide base in the candidate sequence identical with nucleotide in the DmGPCR sequence with specific polynucleotide sequence with after realizing maximal sequence homogeny percentage.

Another aspect of the present invention is to identify other animal with the nucleotide sequence of DmGPCR disclosed herein, comprises the homologue of the DmGPCR in mammal, vertebrate and the invertabrate.Any nucleotide sequence disclosed herein, or its part can be used as for example probe, and with well known to a person skilled in the art screening technique garbled data storehouse or nucleic acid library, for example, homologue is identified in genome or cDNA library.

Polynucleotide sequence information provided by the invention makes that expressing encoded polypeptides on a large scale with well known and technology conventional practice becomes possibility.Polynucleotide of the present invention can also be by the technology of knowing, for example Southern and/or Northern hybridization, and PCR (PCR) identifies and the polynucleotide that separate the relevant DmGPCR polypeptide of coding, for example allele variant and species homologue.The example of related polynucleotides comprises genome sequence, comprises the polynucleotide of the polypeptide of allele variant and coding and DmGPCR homology, and the structurally associated polypeptide of biology, immunity and/or the physical property of total one or more DmGPCR.Also can pass through the gene of the protein of Southern and/or pcr analysis identification code and DmGPCR homology, be used for disease at the GPCR of animal model.Knowledge to the DmGPCR dna sequence dna also can be by expressing the control regulating and controlling sequence by identification code DmGPCR with Southern hybridization or PCR (PCR), and for example the genomic dna sequence of promoter, operon, enhancer, inhibition etc. becomes possibility.Polynucleotide of the present invention also can be used for cross experiment and come identification of cell to express the ability of DmGPCR.Polynucleotide of the present invention also can provide the basis of diagnostic method, are used to identify the ectozoic existence that can express DmGPCR and cause morbid state, and this information is used for diagnosis and selects methods of treatment.

This paper is for the fragment that makes persons skilled in the art can access each possible total length polynucleotide that discloses of the total length polynucleotide of encoding D mGPCR polypeptide.Therefore, the invention provides the polynucleotide passage of encoding D mGPCR, contain at least 14, preferably at least 16,18,20,25, the continuous nucleotide of the polynucleotide of 50 or 75 encoding D mGPCR.Fragment polynucleotide of the present invention can contain the sequence for the polynucleotide sequence uniqueness of encoding D mGPCR, therefore only (i.e. " single-minded ") under highly strict or medium stringent condition with the multi-nucleotide hybrid of encoding D mGPCR (or its fragment).The polynucleotide passage of genome sequence of the present invention is not only the coding region uniqueness, also comprises the sequence derived from introne, regulation and control zone and/or other non-translated sequence.Sequence to polynucleotide uniqueness of the present invention is by more discernible with other known polynucleotide, and can be by using the conventional arrangement contrast program of using in this area, and for example those that can obtain in public's sequence library are identified.These sequences also can be by the identification of Southern hybridization analysis, to determine the number of polynucleotide with the genomic DNA fragment of hybridization.Polynucleotide of the present invention can make the mode mark that it can be detected, comprise radioactivity, fluorescence and enzyme labeling.

The fragment polynucleotide are useful especially as the probe that detects DmGPCR total length or fragment polynucleotide.Can in kit, comprise one or more polynucleotide, be used to detect the existence of the polynucleotide of encoding D mGPCR, or be used to detect the variation of the polynucleotide sequence of encoding D mGPCR.

The present invention also is included under medium strictness or the high stringent condition and SEQ ID NOs:1, and 3,5,7,9,11,13,15,17,19,21, or the DNA of the encoding D mGPCR polypeptide of the noncoding strand of 23 polynucleotide or complementary strand hybridization.

Exemplary high stringent hybridization condition is as follows: hybridize in hybridization solution for 42 ℃, this solution contains 50% formamide, 1%SDS, 1M NaCl, 10% sulfuric acid glucose, and 60 ℃ are washed 2 times in the wash solution that contains 0.1X SSC and 1%SDS.Should understand in the field, can be by changing the condition that temperature and damping fluid or salinity realize equating strict degree, as (volumes .) such as Ausubel, Protocols in Molecular Biology, John Wiley; Sons, 1994, pp.6.0.3-6.4.10 is described.Can determine or according to the length of probe and the variation in guanosine/cytidine (GC) base pairing percent accurate Calculation hybridization conditions by experience.Can be as (volumes) such as Sambrook, MolecularCloning:A Laboratory Manual, Cold Spring Harbor Laboratory Press:Cold SpringHarbor, New York, 1989, the described calculating hybridization conditions of pp.9.47-9.51.

Knowledge with nucleotide sequence information disclosed by the invention, those skilled in the art can originate (being different tissues or different biological) from difference by various well known by persons skilled in the art, or as Sambrook etc., Molecular cloning:a laboratory manual, second edition, Cold Spring Harbor Press, ColdSpring Harbor, NY, 1989 (in this complete introducing for your guidance), the nucleotide sequence of encoding D mGPCR was identified and obtained to described method.

For example, can obtain the DNA of encoding D mGPCR by oligonucleotide probe screening mRNA, cDNA or the genomic DNA that produces with DmGPCR gene order information provided herein.But available detection moiety, for example fluorophor, radioactive atom or chemiluminescent groups, according to method well known by persons skilled in the art and that the conventional hybridization test is used, for example Sambrook etc. is described, label probe.

Contain that the nucleic acid molecules of above-mentioned any DmGPCR nucleotide sequence can choose wantonly with PCR (PCR) method, synthetic with the PCR Oligonucleolide primers that this paper nucleotide sequence produces.See 4,683,202 of the U.S. Patent number 4,683,195 of Mullis etc. and Mullis.The method that the PCR reaction provides selectivity to improve specific nucleic acid sequence concentration is even this sequence is not purified before and only have single copy in a concrete sample.This method can be used for increasing strand or double-stranded DNA.Method relates generally to two kinds of oligonucleotide probes as primer, carries out polymerase-mediated the duplicating that required nucleic acid molecules masterplate relies on.

Those skilled in the art know various optional clones and amplification in vitro method.The example of these technology can be at for example Berger etc., Guide to Molecular Cloning Techniques, Methods in Enzymology152 Academic Press, Inc., find among the San Diego, CA (Berger), for your guidance in this complete introducing.

Nucleic acid molecules of the present invention and its fragment of deriving can be used for screening restriction fragment length polymorphism and gene mapping.

Available robotization sequencing obtains or verifies the nucleotide sequence of DmGPCR.DmGPCR nucleotide of the present invention is 100% accurately.Yet as known in the art, the nucleotide sequence that automatic mode obtains may contain some mistakes.The nucleotide sequence that robotization is determined shows at least about 90% with the accurate nucleotides sequence of given nucleic acid molecules usually, more generally at least about the homogeny of 95%-99.9%.Sequence can be with the more accurate mensuration of manual sequence measurement known in the art accurately.The mistake that causes one or more nucleotide to insert in the sequence or lack can cause translating the middle frame drift, thereby makes that the amino acid sequence of prediction is different with the actual nucleotide sequence that this nucleic acid molecules begins from the mutational site.

Expression constructs and carrier

Also provide the spontaneous replicability recombinant expression construct thing that mixes in the polynucleotide of the present invention, for example plasmid and viral DNA carrier.This paper uses DNA or the RNA of amplification coding DmGPCR and/or expresses the carrier of the DNA of encoding D mGPCR.But carrier of the present invention includes but not limited to plasmid, bacteriophage, clay, episome, virion, virus and dna integration fragment (for example can be integrated into the fragment of host genome by homologous recombination).Virion can include but not limited to adenovirus, baculoviral, parvovirus, herpesviral, poxvirus, the related virus of gland, Smeliki forest virus, vaccinia virus and retrovirus.The example of expression vector includes but not limited to pcDNA3 (Invitrogen) and pSVL (Pharmacia Biotech).Other expression vector includes but not limited to the pSPORT carrier, pGEM carrier (Promega), pPROEX carrier (LTI, Bethesda, MD), Bluescript vector (Stratagene), pQE carrier (Qiagen), pSE420 (Invitrogen), and pYES2 (Invitrogen).

Expression constructs also is provided, and wherein the polynucleotide of encoding D mGPCR are connected with the transcription terminator navigability with endogenous or heterogenous expression control dna sequence dna.Express the control dna sequence dna and generally include promoter, enhancer, operon and regulating element binding site, normally select, wherein utilized expression constructs based on expression system.Usually select promoter and enhancer sequence according to the ability that improves gene expression, and the ability of expressing according to regulatory gene is usually selected operon sequence.But expression constructs of the present invention also can comprise the sequence of one or more selected markers of encoding, thereby can identify the host cell that carries this construction.Expression constructs also can contain the sequence of the homologous recombination in helpful and/or the startup host cell.Construction of the present invention also can contain duplicate necessary sequence in host cell.

Available expression constructs production encoded protein, but also can simply be used for the polynucleotide sequence of amplification coding DmGPCR.In certain embodiments, carrier is an expression vector, and polynucleotide wherein of the present invention are connected with the polynucleotide navigability that contains expression control sequenc.Some expression vectors are reproducible DNA constructions, and wherein the dna sequence dna of encoding D mGPCR is connected with the suitable control sequence navigability that can influence DmGPCR expression in the proper host cell.When navigability between the DNA zone connected or connect, they were correlated with on the function each other.For example, if promoter is connected or connects with the coded sequence navigability, then its control sequence transcribes.Amplification vector does not need to express the control structure territory, and needs and can duplicate in the host, and this is normally given by replication origin and the selection gene that can promote the identification conversion product.The needs of control sequence in the expression vector are looked selected host and method for transformation and different.Usually, control sequence comprises transcripting promoter, the operon sequence that optional control is transcribed, and the sequence with translation termination is transcribed in the sequence of the mRNA ribosomes of encoding suitable combination (site) and control.

Carrier can contain the promoter that can be discerned by host living beings.Promoter sequence of the present invention can be protokaryon, eucaryon or virus.The example of suitable protokaryon sequence comprises PR and PL promoter (Thebacteriophage Lambda, Hershey, the A.D. of phage, compile Cold Spring Harbor Press, Cold SpringHarbor, NY, 1973, in this complete introducing for your guidance; Lambda II, Hendrix, R.W., Ed., ColdSpring Harbor Press, Cold Spring Harbor, NY, 1980, in this complete introducing for your guidance); Colibacillary trp, recA, heat shock and lacZ promoter and SV40 promoter morning (Benoist etc., Nature, 1981,290,304-310, in this complete introducing for your guidance).Other promoter includes but not limited to the promoter of mouse mammary adenoma virus, human immunodeficiency virus's (HIV) the terminal duplicate block of length, maloney leukemia virus, cytomegalovirus immediate early promoter, Epstein-Barr virus, Rous sarcoma virus, human actin, human myoglobulin, human hemoglobin, people's muscle creatine and human metal thioalbumen.

In carrier of the present invention, also can comprise other regulating and controlling sequence.The example of suitable regulating and controlling sequence comprises for example SD sequence of bacteriophage MS-2 rdrp gene and phage gene cII.The SD sequence can cause the expression of ripe DmGPCR albumen directly following the DNA of an encoding D mGPCR thereafter.

In addition, suitable expression vector can comprise suitable mark, thereby can screen through transformed host cells.Know with any those skilled in the art, and at for example Sambrook etc., any in the various technology described in the document of vide ut supra carries out selected host's conversion.

Can pass through carrier construction, comprise the external source starting point or provide replication origin by the host cell chromosome replicanism.If vector integration is gone into host chromosome, the latter is just enough.In addition, those skilled in the art use the carrier that contains the virus replication starting point would rather not, but and by the method transformed mammalian cell with selected marker and DmGPCR DNA cotransformation.An example of appropriate flags is dihyrofolate reductase (DHFR) or thymidine kinase (for example U.S. Patent number 4,399,216).

The nucleotide sequence of encoding D mGPCR can be recombinated according to routine techniques with carrier DNA, comprise that blunt end or staggered end connect, restriction enzyme digests so that suitable end to be provided, and suitably fills cohesive end, alkaline phosphatase treatment to be avoiding unwanted connection, and is connected with suitable ligase.Sambrook etc. disclose these technology, and have been well known in the art on seeing.The method of structure mammalian expression vector such as Okayama etc., Mol.Cell.Biol., 1983,3,280; Cosman etc., Mol.Immunol.1986,23,935; Cosman etc., Nature, 1984,312,768; EP-A-0367566 and WO 91/18982 are described, all in this complete introducing for your guidance.

Host cell

Another aspect of the present invention provides host cell, comprises prokaryotic and eukaryotic, contains polynucleotide of the present invention (or carrier of the present invention) with the form of the DmGPCR expression of polypeptides that allows coding.Polynucleotide of the present invention can be used as cyclic plasmid or contain the linear DNA in protein coding zone of separation or the part of viral vectors, are introduced into host cell.DNA is introduced the method for host cell and know, and conventional use in art technology, comprise conversion, transfection, electroporation, nuclear injection or and carrier, for example fusions such as liposome, micelle, ghost and bioplast.Expression system of the present invention comprises bacterium, yeast, fungi, plant, insect, invertabrate, vertebrate and mammal cell line system.

The invention provides host cell with polynucleotide of the present invention or carrier of the present invention conversion or transfection (stable or instantaneous).As mentioned above, these host cells are used to the polynucleotide that increase, and also are used to express DmGPCR polypeptide or its fragment of this polynucleotide encoding.

Aspects more of the present invention also provide the transformed host cells with expression vector, and this expression vector contains above-mentioned any nucleic acid molecules.When expression vector was introduced into proper host cell, the expression of nucleotide sequence took place.The proper host cell of expressing polypeptide of the present invention includes but not limited to prokaryotic, yeast and eukaryotic.If use prokaryotic expression carrier, proper host cell will be the prokaryotics of the sequence of any energy expression cloning.Suitable prokaryotic includes but not limited to the bacterium of Escherichia coli, bacillus, detection of Salmonella, pseudomonad, streptococcus and staphylococcus.

If use carrier for expression of eukaryon, proper host cell can be any eukaryotics that can express this cloned sequence.Eukaryotic can be the cell of higher eucaryotic cells (animal).Suitable eukaryotic includes but not limited to non-human mammal histocyte and people's tissue culture cells.Host cell includes but not limited to insect cell, HeLa cell, Chinese hamster ovary cell (Chinese hamster ovary celI), African green monkey kidney cell (COS cell), people's 293 cells and mouse 3T3 fibroblast.The propagation of these cells in cell culture is conventional program (see for example Tissue Culture, Academic Press, Kruse and Patterson compile, 1973, in this complete introducing for your guidance).

In addition, available yeast host is as host cell.The example of yeast cells includes but not limited to saccharomyces cerevisiae, Pichia pastoris and Kluyveromyces.The example of yeast host is saccharomyces cerevisiae (S.cerevisiae) and Pichia pastoris (P.pastoris).Yeast vector can contain the replication sequence starting point from the 2T yeast plasmid, spontaneous replication sequence (ARS), promoter region, polyadenylation sequence, but translation termination sequence and selectable marker gene.This paper also comprises the shuttle vector that duplicates in yeast and the Escherichia coli.

In addition, available insect cell is as host cell.In one embodiment, express polypeptide of the present invention with baculovirus expression system and (see Luckow etc., Bio/Technology, 1988,6,47, BaculovirusExpression Vectors:A Laboratory Manual, (volumes) such as O ' Rielly, W.H.Freeman and Company, New York, 1992 and U.S. Patent number 4,879,236, respectively for your guidance) in this complete introducing.In addition, MAXBAC for example ^TMComplete baculovirus expression system (invitrogen) is used in the production in the insect cell.

In another related embodiment, the invention provides the method that produces DmGPCR polypeptide (or its fragment), comprise step: in nutrient medium, cultivate host cell, isolated polypeptide or its variant from cell or nutrient culture media.Because DmGPCR is a kind of 7 transmembrane receptors, should understand in some application, for example in some activity test, separation may relate to and separate the cell membrane that is embedded with this polypeptide, and other may need to separate more completely in using.

Host cell of the present invention is the immunogenic valuable source that exploitation can play the antibody of specific immune response with DmGPCR.Host cell of the present invention also can be used for the method for large-scale production DmGPCR polypeptide, cultured cell in proper culture medium wherein, pass through purification process known in the art from cell or from the nutrient culture media of cellular incubation, for example conventional chromatography, comprise immunoaffinity chromatography, receptor affinity chromatography, hydrophobic interaction chromatography, lectin affinity chromatography, size exclusion chromatography, kation or anion-exchange chromatography, high pressure liquid chromatography (HPLC) (HPLC), reversed-phase HPLC etc., separate required polypeptide product.Other method of purifying comprise wherein express required protein and purifying become have special can be by the fused protein of tail, mark or the chelating molecule of specificity binding partners or reagent identification.Can cut the albumen of this purifying, obtain desired protein, or not handle, still as complete fusion.This cutting process cuts this fusion component, owing to can produce the desirable proteins with extra amino acid residue.

Understanding to the DmGPCR dna sequence dna makes it possible to change cell, thereby allows or improve the expression of endogenous DmGPCR.Can modify cell (for example passing through homologous recombination), replace naturally occurring DmGPCR promoter by or part whole and strengthen expression, thereby make cell express DmGPCR with higher level with all or part of allogeneic promoter.Allogeneic promoter is to insert (seeing for example PCT international publishing WO94/12650, PCT international publishing WO92/20808 and PCT international publishing WO91/09955) with endogenous DmGPCR coded sequence navigability ways of connecting.Also should consider, except allogeneic promoter DNA, also can therewith insert amplifiable marker DNA (as the multi-functional CAD gene of the synzyme of ada, dhfr and encoding carbamoyl phosphate synthase, asparagine transcarbamylase and dihydroora tase), and/or introne DNA.If be connected, the amplification of marker DNA is caused the coamplification of DmGPCR coded sequence in the cell with the Standard Selection method with the DmGPCR coded sequence.

Knock out

Dna sequence dna information provided by the invention also make exploitation (for example, see Capecchi by homologous recombination or " knocking out " strategy, Science, 1989,244,1288-1292) can not expressive function DmGPCR or the individuality of expression DmGPCR variant become possibility.These individualities (especially comprising insect and worm) can be used as the model of the correctives of the activity in vivo of studying DmGPCR and DmGPCR, also can be used for further illustrating the effect of DmGPCR in insect and the worm.

Antisense

The present invention also makes identification and become possibility with the antisense polynucleotides of the multi-nucleotide hybrid of encoding D mGPCR.The antisense polynucleotides of total length and fragment is provided.Fragment antisense molecule of the present invention comprise those specific recognition or can with the sequence (sequence relatively more definite with the DNA of other coding known molecular) of DmGPCR expression control sequenc or DmGPCR RNA hybridization as the DNA of encoding D mGPCR.Evaluation can be by the sequence library of various public Ke De to the sequence of DmGPCR coded polynucleotide uniqueness, and/or is undertaken by commercially available sequence comparison program.After having identified required sequence, separate by restrictive diges-tion or with various arbitrarily polymerase chain reaction technologies well known in the art.Antisense polynucleotides is special closely related to the expression of DmGPCR with the cell of regulating expression DmGPCR mRNA.

The antisensenucleic acids that can combine with DmGPCR expression control sequenc or DmGPCR RNA specificity (preferred 10-20 base pairing oligonucleotides) is introduced cell (for example by viral vectors or micella dispersant system, for example liposome).Antisensenucleic acids is listed in the cell with DmGPCR target nucleotides sequence and combines, and prevents transcribing and/or translating of target sequence.Special consideration D2EHDTPA and methyl-phosphonate antisense oligonucleotides are used for treatment of the present invention.The also available poly-L-Lysine of antisense oligonucleotides, transferrin polylysine or cholesterol molecule are further modified at its 5 ' end.Transcribe or translation skill on suppress DmGPCR and express cell or the animal model that can be used for producing research DmGPCR biological agent.

Derived from the antisense oligonucleotides of the nucleotide sequence of encoding D mGPCR of the present invention or be selected from SEQ ID NOs:1,3,5,7,9,11,13,15,17,19,21, or the fragment of 23 nucleotide sequence, or be used for surveying gene expression at various tissues with its homology or complementary sequence.For example, can survey tissue by conventional planning autography technology with carrying the oligonucleotide probe of surveying group in position.Can be from SEQ ID NOs:1,3,5,7,9,11,13,15,17,19,21, or 23, or corresponding its mRNA, include but not limited to that initiation codon, TATA box, enhancer sequence etc. select the antisense oligonucleotides at the control region of nucleotide sequence.

Transcription factor

The DmGPCR sequence of the present invention instruction makes regulates the new transcription factor of the expression of DmGPCR in n cell and individuality and is transformed by DmGPCR or cells transfected becomes possibility.For example, shown by zinc to refer to that the Cys2-His2 zinc finger protein that functional domain combines with DNA can change by commensurate structure, thereby discerned different target sequences.These artificial zinc finger proteins can play the effect of gene switching with high-affinity and the specific target site of low dissociation constant identification, express with regulatory gene.The present invention makes it possible to known method the understanding of specific DmGPCR target sequence, the modeling and the screening phage display library of for example uniting based on structure come the narrow spectrum zinc finger protein of target sequence is carried out engineered (Segal etc., Proc.Natl.Acad.Sci.USA, 1999,96,2758-2763; Liu etc., Proc.Natl.Acad.Sci.USA, 1997,94,5525-5530; Greisman etc., Science, 1997,275,657-661; Choo etc., J.Mol.Biol., 1997,273,525-532).Each zinc refers to functional domain identification three or more to base usually.Because the recognition sequence of 18 base-pairs is long enough usually, giving its uniqueness to any known group, expection has 6 series connection and repeats zinc finger protein that zinc refer to and can guarantee selectivity (Segal etc.) to particular sequence.Artificial zinc based on the DmGPCR sequences Design refers to that repetitive sequence merges, in activating or suppress activation or inhibit feature territory (Liu etc.) that DmGPCR expresses.Perhaps, zinc can be referred to that functional domain merges by the connector area and the TATA box-binding factor (TBP) of different length between zinc finger peptide and the TBP, and generation transcriptional activation agent or inhibitor (Kim etc., Proc.Natl.Acad.Sci.USA, 1997,94,3616-3620).These albumen and polynucleotides encoding them have the purposes that DmGPCR expresses in the control agent.Can this new transcription factor be passed to target cell or introduce protein by the construction (gene therapy) that transfection can be expressed new transcription factor.Also but the zinc finger protein transformed of project engineering combines with the RNA sequence, is used for the treatment of (McColl etc., Proc.Natl.Acad.Sci.USA, 1997,96,9521-9526 as the alternative method of antisense or catalytic RNA method; Wu etc., Proc.Natl.Acad.Sci.USA, 1995,92,344-348).The present invention has considered to design the method for the zinc finger protein of these transcription factors and customization according to gene order of the present invention, and they can be used for regulating, and DmGPCR expresses in the cell (natural or cell transformed).Hereditary fill-in in these cells contains above-mentioned sequence.

Polypeptide

The present invention also provides the purifying of polynucleotide encoding of the present invention and the DmGPCR polypeptide that separates, comprises having SEQ ID NOs:2, and 4,6,8,10,12,14,16,18,20,22, or the DmGPCR polypeptide of the amino acid sequence of listing in 24.

Should be understood that the outer epi-position of born of the same parents is for producing and screening antibody and other can be particularly useful with the compound of receptors bind such as DmGPCR.Therefore, in another embodiment, the invention provides the extracellular domain (for example one of the terminal extracellular domain of N-and three born of the same parents' outer shrouds) that contains at least one DmGPCR, for example the purifying and the polypeptide that separates of the terminal extracellular domain of the N-of DmGPCR.Also comprise purifying and the polypeptide that separates in the scope of the invention, it contains, and ring connects the terminal cytoplasmic region of C-of membrane-spanning domain, DmGPCR and the DmGPCR fragment of fusions thereof in the membrane-spanning domain that is selected from DmGPCR membrane-spanning domain, DmGPCR born of the same parents' outer shroud and connects, the DmGPCR born of the same parents.These fragments can be the continuous parts of natural receptor.Yet, will be understood that also the knowledge of DmGPCR gene provided herein and protein sequence is able to discontinuous each functional domain in the energy recombinant natural protein.Be called " tmtrestall (and Parodi etc., Comput.Appl.Biosci., 1994,5,527-535) " FORTRAN computer program shows that DmGPCR contains membrane spaning domain.

The present invention also comprises with reference polypeptide of the present invention having at least 99%, at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, and the polypeptide of or at least 50% homogeny and/or homology.Be defined as two sequences with respect to reference amino acid sequence of polypeptide of the present invention " homogeny " percentage this paper and arrange object, introduce breach on demand, realize maximal sequence homogeny number percent, and any conservative property is not replaced be considered as sequence identical after, the percentage of the amino acid residue identical in the candidate sequence with DmGPCR sequence residue.With respect to reference polypeptide of the present invention, amino acid sequence " homology " percentage refers to that at this two sequences is aligned, and introduce breach on demand and realize maximal sequence homogeny number percent, also any conservative replacement is considered as sequence identical after, the percentage of the amino acid residue identical in the candidate sequence with residue in the DmGPCR sequence.

On the one hand, homology percentage is calculated to be when arranging contrast, the identical percentage of amino acid residue in amino acid residue in less one of two sequences and the sequence of comparing, in 100 amino acid lengths, can introduce four breach so that can at utmost aligned (Dayhoff, in Atlas of Protein Sequence andStructure, vol.5, National Biochemical Research Foundation, Washington, D.C., 1972, p.124, be incorporated herein for your guidance).

Can separate from n cell source, or chemosynthesis and produce polypeptide of the present invention by the recombination method that comprises host cell of the present invention.Expectation provides posttranslational modification (for example glycosylation, brachymemma, fatization and phosphorylation) with mammalian host cell, to satisfy the best bioactive needs of recombination expression product of the present invention.The glycosylation of DmGPCR polypeptide and non-glycosylated form belong to the present invention.

The present invention has also comprised DmGPCR polypeptide variants (or congener).In an example, inserting provides the insertion variant, wherein inserts one or more amino acid residues in the DmGPCR amino acid sequence.Embolus can be positioned at the two or an end of protein terminal, maybe can be positioned at DmGPCR amino acid sequence interior zone.Insertion variant with extra residue of one or two for example can comprise endways, fusion or have the albumen of amino acid tail or mark.

Insert variant and comprise the DmGPCR polypeptide, wherein on DmGPCR amino acid sequence or its biological active fragment, add one or more amino acid residues.

Variant product of the present invention also comprises ripe DmGPCR product, has promptly wherein removed leading or burst, and has had the DmGPCR product of extra n terminal residue.Extra n terminal residue can maybe can comprise the one or more residues that can not identify from what concrete protein derived from other protein.-1 DmGPCR product (Met-1-DmGPCR) with extra methionine residues is as-2 and-1 DmGPCR products (Met-2-Lys-1-DmGPCR) with extra methionine and lysine residue in the position in the position in consideration, and the DmGPCR variant with extra Met, Met-Lys, Lys residue (or one or more alkaline residue) is particularly useful for strengthening the recombinant protein production in the bacterial host cell.

The present invention also comprises owing to use specific expression system to cause having the DmGPCR variant of additional amino acid residue.For example, be used as the commercially available carrier that glutathione-S-transferase (GST) fusion part is expressed required polypeptide, provide after downcutting the GST component, at-1 required polypeptide with extra glycine residue from required polypeptide.Also consider to express in other carrier system the variant that obtains.

Insert variant and also comprise fusion, wherein the amino terminal of DmGPCR and/or carboxyl terminal and another polypeptide merge.

On the other hand, the invention provides the disappearance variant, wherein the one or more amino acid residues in the DmGPCR polypeptide are removed.Disappearance can influence one or two end of DmGPCR polypeptide, or removes the one or more non-terminal amino acid residue of DmGPCR.Therefore, the disappearance variant comprises all DmGPCR polypeptide fragments.

The present invention also comprises SEQ ID NOs:2,4,6,8,10,12,14,16,18,20,22, or the polypeptide of sequence fragment of listing in one of 24, wherein these fragments have kept the biology (for example part in conjunction with and/or intracellular signal transmission) and the immunological properties of DmGPCR polypeptide.The present invention considers that any polypeptide as herein described contains at least 5,10,15,20,25,30,35, or the fragment of 40 conservative amino acid.Polypeptide fragment can show the unique or specific antigen performance to DmGPCR and its allele product and species homologue.The method preparation of available any well known and conventional use has the fragment of the present invention of required biology and immunology performance.

In yet another aspect, the invention provides the replacement variant of DmGPCR polypeptide.The those polypeptides that replaces that variant comprises that the amino acid residue of wherein one or more DmGPCR polypeptide is removed and replaced by other residue.In one aspect, replace character and guard, yet the present invention also considers non-conservative replacement.To this, definable for the conservative replacement of this purpose be following table 1,2 or 3 list those.

Variant polypeptide comprises the conservative replacement that those are introduced by the polynucleotide of modifying code book invention polypeptide.Amino acid can be classified according to physical property with to the contribution of secondary and three grades of protein structures.In the art, conservative replacement is considered to the amino acid that has similar quality with a kind of aminoacid replacement another kind.Exemplary conservative replacement (, the 10th page, publishing (PCT/GB96/02197,96/9/6 submits to) on March 13rd, 1997) as described in Table 1 from WO97/09433, as hereinafter:

Table 1

The conservative I that replaces

Side chain

Feature amino acid

Lipophilicity

Nonpolar GAP

ILV

The not charged CSTM of polarity

NQ

The charged DE of polarity

KR

Aromatics HFWY

Other NQDE

In addition, conserved amino acid such as Lehninger, (BIOCHEMISTRY, second edition; Worth Publishers, Inc.NY, NY, 1975, pp.71-77) described grouping is as hereinafter table 2 is listed

Table 2

The conservative II that replaces

Side chain

Feature Amino acid

Nonpolar (hydrophobicity)

A. lipophilicity: ALIVP

B. aromatics: FW

C. sulfur-bearing: M

D. border: G

Not charged-polarity

A. hydroxyl: STY

B. acid amides: NO

C. sulfydryl: C

D. border: G

Positively charged (alkalescence): KRH

Electronegative (acidity): DE

As another kind of selection scheme, exemplary conservative replacement is shown in hereinafter table 3.

Table 3

The conservative III that replaces

The exemplary replacement of original residue

Ala(A)????????????????????????Val，Leu，Ile

Arg(R)????????????????????????Lys，Gln，Asn

Asn(N)????????????Gln，His，Lys，Arg

Asp(D)????????????Glu

Cys(C)????????????Ser

Gln(Q)????????????Asn

Glu(E)????????????Asp

His(H)????????????Asn，Gln，Lys，Arg

Ile(I)????????????Leu，Val，Met，Ala，

Phe，

Leu(L)????????????Ile，Val，Met，Ala，Phe

Lys(K)????????????Arg，Gln，Asn

Met(M)????????????Leu，Phe，Ile

Phe(F)????????????Leu，Val，Ile，Ala

Pro(P)????????????Gly

Ser(S)????????????Thr

Thr(T)????????????Ser

Trp(W)????????????Tyr

Tyr(Y)????????????Trp，Phe，Thr，Ser

Val(V)????????????Ile，Leu，Met，Phe，Ala

The definition that should understand polypeptide of the present invention should comprise the modified polypeptides insertion, disappearance or the replacement of carrying except amino acid residue.For example, modification can be a covalency character, for example comprises with polymkeric substance, lipid, other is organic and the chemical bonding of inorganic group.Can prepare the circulating half-life that these derivants improve polypeptide, maybe can be designed to improve the target ability of polypeptide for required cell, tissue or organ.Similarly, the present invention also comprises through covalent modification, thereby comprises one or more water-soluble polymeric combination, for example comprises the DmGPCR polypeptide of polyglycol, polyoxyethylene glycol or polypropylene glycol.

The part that can show natural DmGPCR is in conjunction with character, and the variant of expressing with higher level, and the variant that the composition active acceptor can be provided, and is particularly useful in test of the present invention; These variants also can be used for test of the present invention, and cell, tissue and animal model are provided, and are used to study unusual DmGPCR activity.

Antibody

The present invention considers that also DmGPCR or its fragment are had specific antibody, for example monoclonal and polyclonal antibody, single-chain antibody, chimeric antibody, difunctional/bispecific antibody, humanized antibody, people's antibody and transplant the antibody of complementary determining region (CDR) comprise the compound that contains CDR sequence that can specific recognition polypeptide of the present invention.The present invention also provides antibody fragment, comprises Fab, Fab ', F (ab ') 2 and Fv.When being used to describe antibody of the present invention, the special identification of term " right ... specificity " expression antibody variable region of the present invention and in conjunction with DmGPCR polypeptide (promptly can distinguish DmGPCR polypeptide and other known GPCR polypeptide) although between DmGPCR and these polypeptide, may have local sequence homogeny, homology or similarity according to the difference of measuring binding affinity.Will be understood that specific antibody also can interact by the sequence in the constant region of outer sequence, particularly molecule of antibody variable region with other protein (for example antibody in staphylococcal protein A or other elisa technique).The shaker test that can determine antibody binding specificity of the present invention is known, and is used by routine in the art.Discussing fully of this test, (volumes) such as visible Harlow, Antibodies ALaboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1988, Chapter 6.Also considered to discern and in conjunction with the antibody of DmGPCR polypeptide fragment of the present invention, condition is that antibody has specificity for the DmGPCR polypeptide.Antibody of the present invention can be known and the conventional method preparation of using in this area with any.

The invention provides DmGPCR of the present invention is had specific antibody.Antibody specificity is as described below.Yet, should emphasize and can produce, and can be considered to " cross reactivity " antibody with the antibody of the irregular cross reaction of DmGPCR (for example because in two peptide species the similar epi-position of irregular existence) from previous described in the literature polypeptide.These cross reacting antibodies are not the antibody to DmGPCR " specificity ".With one of several method, western blot analysis for example well known in the art determines whether a kind of antibody is specific for DmGPCR, or with the known acceptor cross reaction of another kind.In order to identify the cell of expressing DmGPCR and regulate the DmGPCR-ligand-binding activity that the antibody that combines with the outer epi-position specificity of the born of the same parents of DmGPCR is useful.

In one changes, the invention provides monoclonal antibody.The hybridoma that produces these antibody also is a part of the present invention.In another kind changes, the invention provides humanized antibody.Humanized antibody can be used for treating the disease that epizoa causes or the interior therapeutic indication of the patient's condition.

In another changes, the invention provides the cell-free composite that contains polyclonal antibody, wherein at least a antibody is DmGPCR specific antibody of the present invention.The antiserum that separates from animal is an exemplary composition, as a kind of composition, contains the sero-fast antibody component that is suspended in again in water or other thinning agent, excipient or the carrier.

In another related embodiment, the invention provides the DmGPCR specific antibody is had specific anti-idiotype.

As everyone knows, antibody contains less antigen binding domain, and it can separate by chemistry or recombinant technique.These functional domains itself are useful DmGPCR binding molecules, also can merge with toxin or other polypeptide.Therefore, in another embodiment, the invention provides the polypeptide of the fragment that contains the DmGPCR specific antibody, wherein said fragment and polypeptide can combine with DmGPCR.As non-limitative example, the invention provides polypeptide as single-chain antibody, CDR-grafted antibody and humanized antibody.

The non-human antibody can be by any methods known in the art humanization.In a method, inhuman CDR is inserted in people's antibody or in the total antibody framework sequence.Can in this antibody framework, introduce more changeableization then, to regulate affinity or immunogenicity.

Antibody of the present invention for example can be used for therapeutic purposes (by regulating epizoa DmGPCR activity), is used to detect or the diagnostic purpose of detection by quantitative epizoa DmGPCR and the purifying of DmGPCR.The kit that also comprises the antibody of the present invention that is used for any purpose described herein.Usually, kit of the present invention comprises that also antibody has the contrast antigen of immunologic opsonin to it.

The present invention also provides the method for using antibody of the present invention.For example, the invention provides the method for regulating the combination of DmGPCR part, comprise step: under the condition of antibody and receptors bind, make DmGPCR and the specific antibody of DmGPCR is contacted.Antibody of the present invention can be used for to regulate the part combination of DmGPCR, controlling insect colony by insect being used anti-DmGPCR antibody.For example, insect can be selected from fly, fruit bat, tick, flea, louse, flat lice, cockroach and bite fly.

Genetically manipulated

Also can be used for individualities such as insect with the DmGPCR genetically manipulated.Genetically manipulated comprises the negative regulation that recovers DmGPCR activity, DmGPCR overexpression and DmGPCR.The present invention also comprises genetically manipulated, so that the DmGPCR activation recovering that loses owing to function mutation.Suitable cell propagation function DmGPCR gene is carried out in external, original position or body, use carrier, more effective is viral vectors (for example adenovirus, the related virus of gland or retrovirus), or external use physics DNA transfer method (for example liposome or chemical treatment).See for example Anderson, Nature, 1998, suppl.392 (6679), 25-20.For other summary of gene therapy technology, see Friedmann, Science, 1989,244,1275-1281; Verma, Scientific American, 1990,68-84; And Miller, Nature, 1992,357,455-460.Also consider the expression that available genetically manipulated (for example antisense processing) comes negative regulation DmGPCR.As non-limitative example, available genetically manipulated is by knocking out or reduce one or more DmGPCR genes or its fragment control insect colony (seeing context).

Composition

Another aspect of the present invention relates to composition, comprises pesticide and pharmaceutical composition, comprises above-mentioned any nucleic acid molecules or recombinant expression carrier and acceptable carrier or thinning agent.Carrier or thinning agent can be acceptable on the materia medica.Suitable carriers such as Remington ' s Pharmaceutical Sciences A.Osol, latest edition described, this is the normative document of this this area, in this complete introducing for your guidance.The example of these carriers or thinning agent includes but not limited to water, salt solution, ringer ' s solution, glucose solution and 5% human serum albumin.Available liposome and non-aqueous carrier, for example fixing oil.Preparation can be sterilized with common technology.

Also comprise in the scope of the present invention and contain the composition that is formulated in polypeptide of the present invention, polynucleotide or the antibody in the acceptable carrier on the materia medica for example.

The invention provides and contain DmGPCR polynucleotide, DmGPCR polypeptide.Anti--DmGPCR antibody, it has DmGPCR in conjunction with the fragment of activity or the insecticides of part, DmGPCR binding partners or DmGPCR correctives.

Kit and method

The invention still further relates to kit, comprise medicine and pesticide kit.Kit can comprise any above-mentioned nucleic acid molecules, any aforementioned polypeptides, or the antibody that combines of any polypeptide above-mentioned and of the present invention, and negative control.Kit can comprise other composition, for example quantitative reagent of instructions, solid phase carrier, help etc.

Can design kit, be used to detect the expression of polynucleotide or encoded protein matter.For example, can provide the oligonucleotide hybridization kit, comprise a container, the specific oligonucleotide probe and the optional container that positive and negative control and/or instructions are housed of DmGPCR specific DNA is housed.Similarly, can provide the PCR kit, comprising a container, the Auele Specific Primer of DmGPCR specific sequence is housed, DNA and optional is equipped with the container of molecular size mark, the positive and negative control and/or instructions.

Hybridization conditions should be in the presence of other nucleic acid molecules, and the condition of hybridization only takes place with gene.Under stringent hybridization condition, only highly complementary nucleic acid array hybridizing.These conditions can prevent to have the nucleic acid hybridization of 1 or 2 mispairing in 20 continuous nucleotides.These conditions as mentioned above.

The specimen that the present invention is fit to the nuclei acid probe method for example comprises the nucleic acid extractive of cell or cell or biological fluids.The sample that is used for said method can be according to test form, detection method and the tissue that will detect, cell or extract change of properties.The method of nucleic acid extractive of preparation cell is well known in the art, is not difficult to be adapted to obtain the sample with the method therefor compatibility.

In yet another aspect, the invention provides polynucleotide in the test sample, the method that causes disease or unusual diagnostic tool as epizoa, wherein the method comprising the steps of: sample is contacted with nucleic acid probe, this probe has SEQ ID NO:2,4,6 with coding under the cross experiment condition, 8,10,12,14,16,18,20,22, with the target set nucleic acid area hybridization of the polynucleotide of 24 sequence, the nucleotide sequence of the complementary series of this polypeptide of the nucleic acid sequence encoding that described probe contains, its fragment and sequence and fragment; (b) detector probe: the existence of target region hybrid and amount, as the indication of disease.

In addition, can provide immunoassay kit, comprise container, DmGPCR protein-specific antibody and the optional container that positive and negative control and/or instructions are housed are housed in the container.

Also provide to can be used for identifying the DmGPCR binding partners, for example the kit of native ligand or correctives (activator or antagonist).Be used for the treatment of disease or unusual material and may in one or more detect in vitro test to described disease or the unusual wrong corresponding activity of treatment, show positive findings.The material of regulating polypeptide active includes but not limited to antisense oligonucleotides, activator and antagonist, and antibody.

The present invention also provides the method for regulating the part associativity of DmGPCR, comprises step: under the condition of antibody and receptors bind, DmGPCR is contacted with the DmGPCR specific antibody.

The method of induce immune response

The method at the immune response of polypeptide of the present invention is induced in the another aspect of the present invention design in individuality, comprise individuality is used the polypeptide that it measures enough induce immune responses.Amount can be determined by those skilled in the art by decisions such as species, individual sizes.

Identify the method for part

Another aspect of the present invention relates to the method for the compound that evaluation combines with the nucleic acid molecules of DmGPCR or encoding D mGPCR, comprise making DmGPCR or its nucleic acid molecules of encoding contacts with compound, and measure whether compound and DmGPCR or its nucleic acid molecules of encoding combines.In conjunction with determining in conjunction with test by well known to those skilled in the art, include but not limited to gel nigration, Western blotting, radiolabeled competition experiments, based on the expression cloning of bacteriophage, by chromatography be divided into level, co-precipitation, crosslinked, interact and catch/double cross analysis, RNA-analysis of protein, ELISA etc., as Current Protocols in Molecular Biology, John Wiley ﹠amp; Sons, NY, 1999 is described, in this complete introducing for your guidance.The compound that screens comprises (can comprise suspection and DmGPCR or compound that its nucleic acid molecules of encoding combines), but be not limited to that born of the same parents are outer, in the born of the same parents, the compound of biology or chemical origin.

The present invention also provides the test of the compound that evaluation can combine with DmGPCR.One of such test comprises makes the composition that contains DmGPCR contact with the compound of suspection with the DmGPCR combination, and measures the combination between compound and the DmGPCR.In certain embodiments, composition comprises the cell of surface expression DmGPCR.In another kind changes, use the DmGPCR that separates or contain the cell membrane of DmGPCR.Can be direct, for example adopt this combination of compound determination of mark, or available several technology, comprise intracellular signal transmission (or measuring variation that the DmGPCR signal the transmits level) indirect determination of the DmGPCR that this compound of mensuration is induced.

The DmGPCR product of available separation or reorganization.Specific binding molecules is identified or developed to DmGPCR variant or the cell of expressing these products, comprises native ligand and synthetic compound.Binding partners can be used for purifying DmGPCR product, and is used for routine immunization method, and the DmGPCR product in liquid and the tissue sample is detected with quantitative.Binding molecule is for the biologically active of regulating (promptly blockade, suppress or stimulate) DmGPCR, and especially relevant with signal transduction activity is useful especially.

DNA provided by the invention and amino acid sequence information can also identify can with DmGPCR polypeptide or the interactional binding partners compound of polynucleotide.The method of identifying the binding partners compound comprises solution trial, in vitro test, and wherein the DmGPCR polypeptide is fixed and based on the test of cell.The drug candidate and the candidate's pesticide that provide treatment or preventative intervention to express the epizoa related pathologies process of DmGPCR to the evaluation of the binding partners compound of DmGPCR polypeptide.

The present invention includes the pilot system of several evaluation DmGPCR binding partners.In solution trial, method of the present invention comprises step: the DmGPCR polypeptide is contacted with one or more candidate's binding partner compounds and (b) identify the compound combine with the DmGPCR polypeptide.Can be by separating DmGPCR polypeptide/binding partners compound, wherein DmGPCR polypeptide and DmGPCR polypeptide/binding partners compound is separated, identify the compound that combines with the DmGPCR polypeptide.Also consider another additional step in another embodiment of the present invention, i.e. physics, biology and/or the biochemical property of this binding partners compound of signature analysis.In one aspect, use the antibody that DmGPCR polypeptide or candidate's binding partners compound are had immunologic opsonin, separate DmGPCR polypeptide/binding partners compound.

In other embodiments, DmGPCR polypeptide or candidate's binding partners compound contain the mark or the tail that can promote its separation, the method of evaluation binding partners compound of the present invention comprises one by interacting with this mark or tail, separates the step of DmGPCR polypeptide/binding partners compound.The demonstration example of such mark is the polyhistidyl sequence, common about 6 histidine residues, thus can separate the compound of such mark by the nickel chelating.Mark that other is well known and commonly used and tail, for example (Eastman Kodak, Rochester NY), are also contained in the present invention FLAG  tail.Mark of the present invention also include but not limited to radioactive label (125I for example, 35S, 32P, 33P, 3H), fluorescence labeling, chemiluminescent labeling, enzyme labeling and immune labeled.

In some embodiment of in vitro test, method provided by the invention comprises step: immobilized DmGPCR polypeptide is contacted with candidate's binding partners compound and (b) detect combining of candidate compound and DmGPCR polypeptide.In another embodiment, candidate's binding partners compound is immobilized, detects the combination of DmGPCR.Finish immobilization with any method well known in the art, comprise the covalent bond with carrier, pearl or chromatographic resin, and non-covalent high-affinity interacts, for example antibodies, or with Streptavidin/biotin, wherein immobilized compound has biotin molecule.Radioactive label on available (i) on-fixed compound, the (ii) marked price of the fluorescence on the on-fixed compound, the (iii) immunologic opsonin antibody of on-fixed compound, (iv) mark that can the fluorescence excitation carrier on the on-fixed compound, on this carrier, be combined with the detection of immobilized compound realization, the technology of also available other well known and conventional use to combination.

The present invention also provides the test based on cell, to identify the binding partners compound of DmGPCR polypeptide.In one embodiment, the invention provides method, comprise step: the DmGPCR polypeptide that makes at cell surface expression contacts with candidate's binding partners compound, and detects the combination of candidate's binding partners compound to the DmGPCR polypeptide.In another embodiment, detect intracellular Ca2+ stream or other physiological event that comprises that the detection molecules combination causes.

In another embodiment of the present invention, the compound that uses high flux screening DmGPCR to be had appropriate combination affinity.Say that simply synthetic a large amount of different little peptide test compounds on solid phase carrier are as the free cpds that is dissolved in the suitable buffer.The peptide test compounds contacts with DmGPCR, washing.Detect the DmGPCR of combination then with method well known in the art.Also can be directly the polypeptide of purifying of the present invention be wrapped on the flat board, be used for above-mentioned in conjunction with test.In addition, available non-neutral antibody capture protein is fixed on the solid phase carrier.

Usually, the DmGPCR of available expression carries out combining test with the HTS of its part of determining.Radioactive isotope with suitable includes but not limited to 125I, 3H, 35S or 32P, the polypeptide that identifies with method mark well known to those skilled in the art.In addition, available well-known process is with suitable fluorescent derivative mark peptide (Baindur etc., Drug Dev.Res., 1994,33,373-398; Rogers, Drug Discovery Today, 1997,2,156-160).One of available several standard methods, comprise and filter acceptor one ligand complex, never isolate the part of combination in the binding partner, detection of radioactive labels in the HTS test, it combines (Williams, Med.Res.Rev., 1991 with receptor-specific in the resulting film preparation thing of the clone of express recombinant protein, 11,147-184; Sweetnam etc., J.Natural Products, 1993,56,441-455).Another kind method comprises scintillation proximity assay (SPA) or FlshPlate form, and wherein these separation are not necessary (Nakayama, Curr.OpinionDrug Disc.Dev., 1998,1,85-91 Boss é etc., J.Biomolecular Screening, 1998,3,285-292).Can in all sorts of ways, comprise that fluorescence energy transfer (FRET), the directly part of fluorophotometric analysis combination, or fluorescence polarization detect combination (Rogers, Drug Discovery Today, 1997,2, the 156-160 of fluorescent ligand; Hill, Curr.Opinion Drug Disc.Dev., 1998,1,92-97).

The ligands specific of DmGPCR is identified in available other test, comprise by of the direct test that combine evaluation target protein part of mensuration test ligand with target protein, with with ion EFI mass spectrophotometry/HPLC method, or other physics and analytic approach are identified the part by the target protein of affinity ultrafiltration.In addition, with (Nature, 1989 such as Fields, 340,245-246) and (Trends in Genetics, 1994 such as Fields, 10,286-292) described yeast two-hybrid system is assessed these combinations indirectly, and these documents all in this complete introducing for your guidance.Two-hybrid system is to be used to detect interactional genetic method between two kinds of protein or the polypeptide.Can be used for identifying the protein that combines with interested known protein, or be used for being demarcated in interactional key function territory or residue.Developed the various variations of this method, be used for the gene of clones coding DNA synthetic proteins, the peptide of evaluation and protein bound, or screening of medicaments.Two-hybrid system utilizes pair of interacting protein, and the transcription activating functional domain is taken to near upstream reporter activation sequences (UAS) DNA combines the territory, carries out in yeast usually.Testing needs to make up two hybrid genes, and coding (1) combines territory and (2) and second kind of activation domain that protein merges with the DNA-that first kind of protein merges.DNA-in conjunction with the territory with first kind of hybridization protein belt to the UAS place of reporter; Yet because most of potein deficiency activation domain, this DNA-does not activate transcribing of reporter in conjunction with hybridization albumen.The second hybridization albumen itself that contains activation domain can not activate the expression of reporter, because it does not combine with UAS.Yet when having two kinds of hybridization protein, the noncovalent interaction of first and second kinds of protein couples together activation domain and UAS, activates transcribing of reporter.For example, when first kind of protein is DmGPCR gene outcome or its fragment, promptly know its can with another kind of protein or nucleic acid interaction, this test will be used to detect the material that can disturb this binding interactions.When in this system, adding different test substances, the expression of monitoring reporter.The existence of inhibitor causes lacking the reporter signal.

When the function of not knowing the DmGPCR gene outcome, when not knowing with part that gene outcome combines, the yeast two-hybrid test also can be used for identifying the protein that combines with gene outcome.In the test of the protein that a kind of evaluation combines with DmGPCR acceptor or its fragment, can use encoding D mGPCR acceptor (or fragment) and UAS fusion polynucleotides in conjunction with territory (i.e. first kind of albumen).In addition, produce in the method and the hybrid gene that has screened the second kind of different protein of encoding respectively that many and activation domain merge.Usually, one or more members of merging the library by total cDNA or genomic DNA second albumen of encoding, each second protein-coding region and activation domain fusion.This system can be used for numerous protein, does not even need to know second protein-bonded identity or the function.System is super-sensitive, can detect the interaction that other method can not disclose; Even snap can trigger and transcribe, and can repeat to be translated to produce, and obtains reporting the stable mRNA of albumen.

The material that available other method search combines with target protein.U.S. Patent number 5,585,277 (being incorporated herein for your guidance) have been described one of direct screening technique that combines of characterization test part and target protein.This method is the potpourri of and non-folded state folding according to protein conduct always, and constantly switches the principle of existence between two states.When test ligand combines with the folded form of target protein (when test ligand is the part of target protein), the protein target molecule that combines with part maintains its folded state.Therefore, with test ligand that target protein combines in the presence of, when folding target protein exists than no part for many.Available any can distinguish the folding of target protein and not the method for folded state determine combining of part and target protein.In order to carry out this test, do not need to know the function of target protein.In fact can include but not limited to metal, polypeptide, protein, lipid, polysaccharide, polynucleotide and little organic molecule by any material of this method assessment as test ligand.

The method of another kind of evaluation target protein part such as Wieboldt etc. (Anal.Chem., 1997,69,1683-1691) described.This technology can a screening arrive the 20-30 kind material that can combine with target protein in combinatorial libraries solution.By simple film washing, the material that combines with target protein can be separated with other library component.The molecule of staying subsequently on the film of selecting especially discharges from target protein, analyzes with HPLC and auxiliary EFI (ionspray) the ion massspectrum method of pressure gas.This method selects target protein is had the library component of maximum affinity, and is useful especially for the micromolecule library.

Other embodiments of the invention comprise uses competitive shaker test, wherein can compete combining polypeptide with test compounds specifically in conjunction with the neutrality antibody of polypeptide of the present invention.With this, available antibodies detects the polypeptide that any and DmGPCR have one or more same antigen determinants.Radiolabeled competitive in conjunction with research as A.H.Lin etc. (Antimicrobial Agents and Chemotherapy, 1997,41 (10), 2127-2131) described, in this complete introducing for your guidance.

Identify the method for correctives

The present invention also provides the method for identifying the correctives of the combination between DmGPCR and the DmGPCR binding partners, comprise step: (a) make DmGPCR binding partners and the composition that contains DmGPCR, under the condition of existence and the correctives compound that does not have supposition, contact; (b) combination between detection binding partners and the DmGPCR; (c) combination in the presence of the supposition correctives according to binding partners and DmGPCR reduces or increases than the combination under the correctives that does not have supposition, identifies the adjusting compound or the correctives compound of supposition.

Transmit (for example) under the not enough situation at the DmGPCR signal, can stimulate the DmGPCR binding partners of DmGPCR activity to can be used as activator because DmGPCR ligand activity deficiency.Transmit under the excessive situation at the DmGPCR signal, the DmGPCR binding partners that seals ligand-mediated DmGPCR signal transmission can be used as the antagonist of DmGPCR.In addition, DmGPCR correctives and DmGPCR polynucleotide and polypeptide can be used for using in the diagnostic test of the caused disease of epizoa or DmGPCR increased activity or damage disease.

In yet another aspect, the invention provides the method for treatment disease or unusual condition, comprise the individuality of this treatment of needs used to regulate to have the SEQ of being selected from ID NO:2,4,6,8,10,12,14,16,18,20,22, or the material of the active or expression of 24 polypeptide of sequence.

Another aspect of the present invention can be regulated expression of DmGPCR polynucleotide or active binding partners or correctives by insect is used, and the method for control insect colony is provided.

Can identify adjusting (promptly strengthen, descend or blockade) DmGPCR activity or the material of expressing by the correctives of cultivating correctives of inferring and the cell that contains DmGPCR polypeptide or polynucleotide and mensuration supposition effect active to DmGPCR or that express.To the effect of DmGPCR with to the effect of other GPCR compound, can assess the selectivity that this compound is regulated the DmGPCR activity by certain compound relatively.Selective modulator can comprise antibody and other protein, polypeptide or the organic molecule that for example can combine with the nucleic acid specificity of DmGPCR polypeptide or encoding D mGPCR.The correctives of DmGPCR activity should be to be used for the treatment of in treatment and active diseases associated of normal or unusual DmGPCR and physiological situation.

DmGPCR polynucleotide and polypeptide, and the DmGPCR correctives can be used for disease that epizoa causes or to strengthen or impaired GPCR activity is the diagnostic test of the patient's condition of feature.

The present invention identifies that the method for correctives comprises the variation of above-mentioned any method, and to identify the binding partners compound, variant comprises following technology, wherein identifies a kind of binding partners, carries out the combination test under the situation that has and do not exist candidate modulator.Under the existence that is combined in candidate modulator between DmGPCR polypeptide and the binding partners compound, relatively, when changing to some extent, be accredited as correctives under the situation non-existent with it.The correctives that improves the combination between DmGPCR polypeptide and the binding partners compound is called as reinforcing agent or activator, and the correctives that reduces the combination between DmGPCR polypeptide and the binding partners compound is called as inhibitor.

The present invention also comprises high flux screening (HTS) test, identifies influence or suppresses the compound of DmGPCR polypeptide biologically active (promptly influence enzymatic activity, in conjunction with activity etc.).The HTS test can be screened a large amount of compounds with effective means.HTS system based on cell is used to investigate the DmGPCR receptor-ligand binding." hitting " or " lead compound " that design HTS test has required character with evaluation then can be from its design variation, to improve required character." hit " or the chemical modification of " lead compound " often based on appraisable structure/activity relationship between " hitting " and the DmGPCR polypeptide.

Correctives in the scope of the invention includes but not limited to non-peptide molecule (for example non-peptide mimics), non-peptide allosteric effector and peptide.The DmGPCR polypeptide or the polynucleotide that are used for this test can be to be free in solution, are fixed on the solid phase carrier, are carried on cell surface or are positioned at cell, or combine with the part of cell.Those skilled in the art can, for example, measure DmGPCR and the compound that will test between the formation of compound.In addition, those skilled in the art can also detect between DmGPCR and its substrate because the minimizing that the compound that the compound that will test causes forms.

Another aspect of the present invention relates to the method for identifying the compound of regulating (promptly increase or reduce) DmGPCR activity, comprises DmGPCR is contacted with compound, measures the activity whether compound changes DmGPCR.Relatively in the activity in the presence of the test compounds and there is not activity under the situation of test compounds.The specific activity that contains the sample of test compounds lacks active high in the sample of test compounds, and compound should improve activity.Similarly, the specific activity that contains the sample of test compounds lacks active low in the sample of test compounds, and compound should suppress activity.

The present invention passes through to use the DmGPCR screening compounds especially in various activity tests.The compound that screens comprises (can comprise compound that suspect to regulate the DmGPCR activity), but is not limited to, born of the same parents are outer, in the born of the same parents, the compound of biology or chemical origin.The DmGPCR polypeptide of Shi Yonging can be any form in these trials, for example is free in the solution, is fixed on the solid phase carrier, is carried on cell surface or is positioned at cell.Those skilled in the art can, for example, measure DmGPCR and the compound that will test between the formation of compound.In addition, those skilled in the art can also detect between DmGPCR and its substrate because the minimizing of the compound of the formation that the compound that will test causes.

The activity of DmGPCR polypeptide of the present invention can be by for example combining with the peptide part of chemosynthesis or being measured by the ability of its activation.In addition, can measure the activity of DmGPCR by measuring its ability that combines with calcium ion, hormone, chemotactic factor (CF), neuropeptide, neurotransmitter, nucleotide, fat, odorant and photon.In addition, the activity of DmGPCR can include but not limited to that the activity of adenyl cyclase, phosphatidase and ion channel is measured the DmGPCR activity by measuring the effector molecules molecule.Therefore, the correctives of DmGPCR activity can change the DmGPCR function of receptors, for example acceptor in conjunction with character or activity, protein mediated signal transduction of G or film location for example.In the various embodiments of method, test can adopt ion flow test, yeast growth test, non-water-disintegrable GTP test (for example [ ³⁵S] GTP γ S test), Ca in the cAMP test, InsP3 test, DG test, aequorin test, luciferase test, born of the same parents ²⁺The FLIPR test of concentration, mitosis test, map kinase activity test, arachidonic acid release test (for example use [ ³H]-arachidonic acid) and the extracellular acidification speed trial, and the test form based on combination or function of other DmGPCR activity known in the art.In these embodiments of the present invention, some comprises any G albumen known in the art, for example G ₁₆, G ₁₅, or chimeric G _Qi5, G _Qs5, G _Qo5, G _Qz5Deng.Method used in the available FaRP activity test well known to those skilled in the art is measured the DmGPCR activity.The biologically active of DmGPCR acceptor of the present invention includes but not limited to and the combining of natural or non-natural part, and any functional activity of GPCR known in the art.The non-limitative example of GPCR activity comprises various forms of transmembrane signal transmission, can relate to the G protein combination and/or to the protein bound influence of the G of various guanylic acids; The exemplary activity of another kind of GPCR is auxilin or the polypeptide combination different with known G protein.

Correctives of the present invention has shown various chemical constitutions, can be categorized as usually natural DmGPCR receptors ligand non-peptide analoglike thing, DmGPCR acceptor peptide and non-peptide allosteric effector and can be used as the activator of DmGPCR acceptor or inhibitor (competitive, anti-competitiveness and noncompetitive) (for example antibody product).The present invention does not limit the source of suitable adjustable agent, can be available from natural origin, and for example plant, animal or mineral extract, or non-natural source, micromolecule library for example comprises the combinational chemistry product and the peptide library of library construction.The example of the peptide modulators of DmGPCR acceptor shows following primary structure: GLGPRPLRF acid amides (SEQ ID NO:49), GNSFLRF acid amides (SEQ ID NO:136), GGPQGPLRF acid amides (SEQ ID NO:102), GPSGPLRF acid amides (SEQ ID NO:103), PDVDHVFLRF acid amides (SEQ ID NO:150), and Jiao-EDVDHVFLRF acid amides (SEQ ID NO:167).

Other test of available detection enzymatic activity includes but not limited to photometric analysis, radiometric analysis, HPLC, galvanochemistry etc., as ENZYME ASSAYS:A PRACTICAL APPROACH, R.Eisenthal and M.J.Danson compile, 1992, Oxford University Press is described, in this complete introducing for your guidance.

The purposes of the cDNA of coding GPCR is known in activity test, and the test method that can test thousands of unknown compounds in high flux screening (HTS) every day has detailed record.Be full of in the document at HTS in conjunction with using the example of radiolabeled part developing drugs (to see Williams, Medicinal ResearchReviews, 1991,11,147-184 in testing; Sweetnam, etc., J.Natural Products, 1993,56, the summary of 441-455).For combination test HTS, recombinant receptor is preferred, because they have better specificity (higher relative purity), can produce a large amount of receptive material, and can use with various forms and (see Hodgson, Bio/Technology, 1992,10,973-980, in this complete introducing for your guidance).

The functional expression of the recombinant receptor that various allos system is known for those skilled in the art is feasible.These systems comprise bacterium, and (Strosberg is etc., Trends in Pharmacological Sciences, 1992,13,95-98), yeast (Pausch, Trends in Biotechnology, 1997,15,487-494), several insect cells (Vanden Broeck, Int.Rev.Cytology, 1996,164,189-268), amphibian cell (Jayawickreme etc., Curr.Opin.Biotechnol., 1997,8,629-634) with several mammal cell line (CHO, HEK293, COS, etc.; See Gerhardt, etc., Eur.J.Pharmacology, 1997,334,1-23).These examples are not got rid of and are used other possible cell expression system, comprise the clone that obtains from nematode (the PCT application, WO98/37177).

In some embodiments of the invention, the method that screening can be regulated the compound of DmGPCR activity comprises makes test compounds contact with DmGPCR, and analyzes the existence of the compound that forms between this compound and the DmGPCR.In such test, common tagged ligand.After suitably cultivating, the combining form of free ligand and existence is separated, dissociate or not the amount of compound token be the measurement index of specific compound and DmGPCR binding ability.

The activation of knowing heteroreceptor in recombination system can cause various biological respinses, and they are protein mediated by the G that expresses in the host cell.The GDP that activator occupies combination on the binding site that GPCR can cause G α subunit is transformed into GTP; The radioactivity of available GTP, non-water-disintegrable derivant [ ³⁵S] GTP γ S measure the combining of activator and acceptor (Sim etc., Neuroreport, 1996,7,729-733).Also available this combination is by in the presence of known activator, and the minimizing of [35S] GTP γ S combination mensuration antagonist combines with acceptor.Therefore, the HTS that can make up based on [35S] GTP γ S combination tests.

The required G albumen of functional expression allos GPCR can be the natural component of host cell, maybe can introduce by the recombinant technique of knowing.G albumen can be complete or chimeric.Usually, can utilize near ubiquitous competitive G albumen (G for example _{α 16}) any acceptor that provides and detectable response pathway are interrelated.G albumen activates and to cause stimulating or suppressing other native protein, the activity that may get in touch with the reacting phase that can measure.

The example of these biological respinses includes but not limited to following: the neutralization of engineered especially yeast cells do not exist the ability of surviving under the limit nutrients situation (Pausch, Trends in Biotechnology, 1997,15,487-494); Measure Ca in the born of the same parents as fluorescent dye ²⁺The change of concentration (Murphy, etc., Curr.Opin.Drug Disc.Dev., 1998,1,192-199).The variation of pH in membrane potential that also available change in fluorescence monitoring part is induced or the born of the same parents; For this purpose, described suitable HTS automated system (Schroeder, etc., J.Biomolecular Screening, 1996,1,75-80).Be presented at the ligand dependent variation of pigment tissue structure in the reaction that heterologous GPCR is activated with the melanocyte of Africa xenopus (Xenopus laevis) preparation; This reaction can adapt to the HTS form (Jayawickreme, etc., Curr.Opin.Biotechnol., 1997,8,629-634).Also can carry out common second messenger (comprising cAMP, phosphoinositide and arachidonic acid) measures.

Use the HTS method of these acceptors to comprise the Chinese hamster ovary celI of permanent transfection, wherein can change in the film of these cell preparation by specificity [ ³⁵S] ability of GTP γ S combination identifies activator and antagonist.In another embodiment of the present invention, the Chinese hamster ovary celI of permanent transfection can be used for preparing the film that contains remarkable quantity recombinant receptor albumen; These film preparation things can be used for using the receptor binding assays of this special receptor specificity radio-labeled part then.In addition, can be used for the function test of HTS, for example fluorescence monitoring contains in the permanent transfection CHO cell of one of these acceptors or acceptor combination Ca in the born of the same parents ²⁺The variation that concentration or membrane potential part are induced.The equally usefully mammalian cell of another kind of form, for example similar form of HEK293 or COS cell.In the HTS form well known to those skilled in the art, the insect cell line of permanent transfection, for example fruit bat S2 cell, with the recombinant yeast cell of expressing the fruit bat acceptor (Pausch for example, Trends in Biotechnology, 1997,15,487-494) also can be used for the present invention.

The present invention considers multiple screening and identifies the part binding inhibitors of DmGPCR acceptor.In an example, the DmGPCR acceptor is fixed, and has and do not exist candidate modulator, for example interaction of assessment DmGPCR and binding partner under the situation of inhibitor compound.In solution trial, there is and does not exist the interaction of analyzing under the situation of candidate inhibitor compound between DmGPCR acceptor and its binding partner in another example.In two kinds of tests, identify the compound that combines that can reduce between DmGPCR acceptor and its binding partners as its inhibitor.The test that another kind is considered relates to the variation of double cross test, wherein identifies the inhibitor of albumen/protein-interacting by positive signal in the host cell that detects conversion or transfection, and as described in PCT publication WO95/20652, publish August 3 nineteen ninety-five.

The candidate modulator that the present invention considers comprises the compound that is selected from possible activator or possible inhibitor library.Many different libraries are arranged, can be used for identifying small-molecule modulators, comprise (1) chemical library, (2) natural product libraries and (3) comprise the combinatorial libraries of peptide, oligonucleotides or organic molecule at random.The chemistry library comprises chemical constitution at random, wherein some are the analog of known compound or the analog that is accredited as " hitting " or " guide " compound in other medicines exploitation screening, some of them also have some to produce from non-directional synthetic organic chemistry material derived from natural products.Natural product libraries is microorganism, animal, plant or the halobiontic set that is used to set up the screening potpourri, this library can produce meat soup and extracts by (1) fermentation with from soil, plant or marine microorganism, or (2) plant or halobiontic extraction are set up.Natural product libraries comprises poly ketonic compound, non-ribosomal peptides and (non-natural takes place) variant thereof.Combinatorial libraries comprises a large amount of peptides, oligonucleotides or organic compound, as potpourri.These libraries are easier to prepare with traditional automatic synthesis method, PCR, clone or patent synthetic method.Interested especially is non-peptide combinatorial libraries.Other interested library comprises peptide, protein, peptide mimics, how parallel synthetic set, reorganization and polypeptide libraries.For combinatorial chemistry with from the summary in the library of its foundation, see Myers, Curr.Opin.Biotechnol., 1997,8,701-707.Optimize the ability that " hitting thing " regulates activity with " hitting thing " (or " guide's thing ") that various libraries evaluation correctivess as herein described can change the candidate.

Can design other candidate inhibitor that the present invention considers, comprise the soluble form of binding partners, and as the binding partners of chimeric or fusion." binding partners " used herein broadly comprises non-peptide modulators, and the peptide modulators of the neuropeptide except native ligand, antibody, antibody fragment and comprise the compound of modification that the DmGPCR expression of gene product that identifies is had the antibody structure territory of immunologic opsonin.

In other embodiments of the invention, polypeptide of the present invention is used as the research tool of evaluation, signature analysis and the purifying of interaction correctives.In polypeptide of the present invention, mix suitable mark by various methods known in the art, catch the functionality molecule with polypeptide.For example, molecule can be cultivated with the polypeptide of mark, and unconjugated polypeptide, quantitative measurement polypeptide complex are removed in washing.The value of quantity, affinity and combination that the data computation polypeptide that obtains with the polypeptide of variable concentrations combines with albumen composition.

The polypeptide of mark also can be used as the reagent of the molecule that this polypeptide of purifying reacts to each other with it, and this molecule includes but not limited to inhibitor.In an embodiment of affinity purification, polypeptide and chromatographic column covalent coupling.Extract cell and film thereof, various cell subfractions pass through post.Molecule combines with the affinity of polypeptide by it with post.Reclaim polypeptide complex from post, dissociate and the molecule that reclaims is carried out protein sequencing.This amino acid sequence is used to identify capture molecules then, or is used to design the oligonucleotides from suitable cDNA library clone corresponding gene.

In addition, can identify to show with DmGPCR part of the present invention to have similar character, but less, and the compound show more than the endogenous ligands in the human or animal body long half-lift.When the design organic compound, molecule of the present invention can be used as " guide " compound.Design is learned the analogies of going up activated compound to known drug, is the method for knowing in the medicine of exploitation based on these " guide " compounds.Usually design, synthesize and test the destination properties of avoiding in a large amount of molecules of random screening with analogies.In addition, the structured data that obtains by the putative amino acid sequence of analyzing dna encoding of the present invention also can be used for having more specific new drug, thereby makes it have higher medicine ability.

Sequence in protein sequence more of the present invention and all available database shows that protein of the present invention and g protein coupled receptor are striden membrane portions remarkable homology.Therefore, the available computers modeling develops the tertiary structure that protein of the present invention is inferred according to the information of available other protein membrane-spanning domain.Therefore, can design novel part based on the structure of the prediction of DmGPCR.

In a specific embodiment, the novel molecule of identifying with screening technique of the present invention is a low molecular weight organic molecules, oral composition or the pharmaceutical composition of wherein available its preparation, for example tablet.Can prepare what conventional administering mode, include but not limited in oral cavity, intravenous, skin, subcutaneous, nose, the muscle or the composition or the pharmaceutical composition of administration in the peritonaeum, comprise nucleic acid molecules, carrier, polypeptide, antibody and compound with screening technique evaluation as herein described.The character of carrier or other composition depends on concrete method of administration and the specific embodiment of the present invention to be administered.Be used for visible Remington ' s PharmaceuticalSciences such as the technology of this situation and the example of scheme, Osol, A (ed.), 1980, in this complete introducing for your guidance.

The dosage of these low molecular weight compounds can be by the morbid state that will treat and the patient's condition, and other clinical factor, for example body weight of the individuality that will treat or situation, and the decision of the approach of administered compound.For the treatment animal, can use the compound of about 0.5mg/kg body weight-500mg/kg body weight.Treatment is normally carrying out than low dosage, and proceeds to and observe required treatment results.

Determine the dosage of compound that individuality is used and the U.S. Provisional Patent Application No.08/702 that the method for the pattern of biological administered compound was submitted to as on August 23rd, 1996,282, described with the international monopoly publication number WO96/22976 that on August 1st, 1996 published, in this complete introducing for your guidance, comprise any accompanying drawing or form.It will be understood by those skilled in the art that these descriptions are applicable to that the present invention also can be easily to its adaptation.

Correct dosage depends on various factors, for example the type of the disease that will treat, the concrete composition of use and individual size and physiological status.Treatment effective dose as herein described can be estimated by cell culture and animal model at first.For example, can in animal model, prepare dosage, reach the circulation composition scope of the IC50 that initial consideration measured in the cell culture test.

Also can determine the plasma half-life and the bio distribution in blood plasma, tumour and major organs of medicine and metabolin, so that select the medicine of a kind of disease of the most suitable inhibition.Can carry out such mensuration, for example, can carry out HPLC to the blood plasma of the animal that heals with medicine and analyze, also available detection method, for example X-ray, cat scan and MRI measure the position of radiolabeled compound.Can in shaker test, show strong inhibitory activity by changing chemical constitution and test again, optimizing, but the compound of pharmacokinetic properties difference.To this, the compound of the good pharmacokinetic characteristic of available demonstration is as model.

Also can carry out toxicity research by measuring the haemocyte composition.For example, can be in suitable animal model the following toxicity research that carries out: 1) to mouse administered compound (also should use and not treat control mice); 2) in each treatment group, regularly obtain blood sample by mouse tail vein; With 3) red blood cell and the white blood cell count of analytic sample, haemocyte form and lymphocyte to the percentage of polymorphonuclear cell.Comparative result to each dosage regimen and contrast will show whether there is toxicity.

When each toxicity research finishes, can pass through kill animals (preferably reports according to the U.S. veterinary science federation guide of U.S. veterinary science federation: Panel on Euthanasia, J.Amer.Vet.Med.Assoc., 1993,202, on the 229-249) further study.Then can be by visual inspection autopsy, check the positive evidence of transfer in the representative animal of each treatment group, abnormal diseases or toxicity.Macroscopic unusual in the record organization done histological examination to tissue.

Compounds and methods for of the present invention, comprise nucleic acid molecules, polypeptide, antibody, compound that screening technique as herein described identifies, have various materia medica and agricultural (for example pesticide) purposes, and can be used for the patient's condition of for example treating or preventing epizoa to cause, or control insect colony.

The present invention also is included in the individuality method of the relevant activity of exciting (stimulations) or the natural binding partners of antagonism DmGPCR, comprises activator or the antagonist of described individuality being used one of enough aforementioned polypeptides that this excitement or antagonism are worked of its amount.One embodiment of the present of invention are the activator of a kind of usefulness protein of the present invention or the disease that an antagonist for treating vermin causes or the method for the patient's condition, comprise individuality is used activator or the antagonist that it measures enough excitements or antagonism epizoa DmGPCR function associated.

Hereinafter table 4 has comprised polynucleotide of the present invention and polypeptide.

Table 4

DmGPCR1DNA ( SEQ ID NO：1 ) ： ATGGCCAACTTAAGCTGGCTGAGCACCATCACCACCACCTCCTCCTCCATCAGCACCAGC CAGCTGCCATTGGTCAGCACAACCAACTGGAGCCTAACGTCGCCGGGAACTACTAGCGCT ATCTTGGCGGATGTGGCTGCATCGGATGAGGATAGGAGCGGCGGGATCATTCACAACCAG TTCGTGCAAATCTTCTTCTACGTCCTGTACGCCACGGTCTTTGTCCTGGGTGTCTTCGGA AATGTCCTGGTTTGCTACGTAGTTCTGAGGAATCGGGCCATGCAGACTGTGACCAATATA TTCATCACGAATCTGGCCCTGTCGGACATATTGCTCTGCGTCCTGGCGGTGCCATTTACT CCGCTTTACACGTTCATGGGTCGCTGGGCCTTCGGCAGGAGTCTGTGCCATCTGGTGTCC TTTGCCCAGGGATGCAGCATCTACATATCCACGCTGACCCTCACCTCGATTGCCATCGAT CGGTACTTCGTTATCATATACCCCTTCCATCCGCGCATGAAGCTCTCCACCTGCATCGGG ATCATAGTGAGCATCTGGGTGATAGCCCTGCTGGCCACCGTTCCCTACGGCATGTACATG AAGATGACCAACGAGCTGGTGAACGGAACGCAGACAGGCAACGAGACCCTGGTGGAGGCC ACTCTAATGCTAAACGGAAGCTTTGTGGCCCAGGGATCAGGATTCATCGAGGCGCCGGAC TCTACCTCGGCCACCCAGGCCTATATGCAGGTGATGACCGCCGGATCAACGGGACCGGAG ATGCCCTATGTGCGGGTGTACTGCGAGGAGAACTGGCCATCGGAGCAGTACCGGAAGGTG TTCGGTGCCATCACAACCACTCTGCAGTTTGTGCTGCCCTTCTTCATCATCTCGATTTGC TACGTGTGGATATCGGTGAAGCTAAACCAGCGGGCCAGGGCCAAGCCGGGATCGAAATCC TCGAGACGGGAGGAGGCGGATCGGGATCGCAAGAAGCGCACCAACCGCATGCTCATCGCC ATGGTGGCGGTATTCGGACTCAGCTGGCTGCCCATCAATGTGGTCAACATATTCGATGAC TTCGATGACAAGTCCAACGAGTGGCGCTTCTACATCCTATTCTTCTTTGTGGCCCACTCT ATTGCCATGAGCTCCACCTGCTACAATCCCTTCCTGTACGCCTGGCTGAACGAGAACTTC CGCAAGGAGTTCAAGCACGTGCTGCCCTGCTTTAATCCCTCGAACAACAACATCATCAAC ATCACCAGGGGCTATAATCGGAGTGATCGGAACACCTGTGGTCCGCGACTGCATCATGGC AAGGGGGATGGTGGCATGGGCGGTGGCAGTCTGGACGCCGACGACCAGGACGAGAACGGC ATCACCCAGGAGACCTGTCTGCCCAAGGAGAAGCTGCTGATTATCCCCAGGGAGCCGACT

TACGGCAATGGCACGGGTGCCGTGTCGCCAATCCTTAGCGGGCGCGGCATTAACGCCGCC CTGGTGCACGGTGGCGACCATCAGATGCACCAGCTGCAGCCGTCACACCATCAACAGGTG GAGCTGACGAGGCGAATCCGCCGGCGGACAGACGAGACGGACGGGGATTACCTGGACTCC GGCGACGAGCAGACCGTGGAGGTGCGCTTCAGCGAGACGCCGTTCGTCAGCACGGATAAT ACCACCGGGATCAGCATTCTGGAGACGAGTACGAGTCACTGCCAGGACTCGGATGTGATG GTCGAGCTGGGCGAGGCAATCGGCGCCGGTGGTGGGGCAGAGCTGGGGAGGCGAATCAAC TGA ( SEQ ID NO：2 ) SEQ ID NO：1DNA ： MANLSWLSTITTTSSSISTSQLPLVSTTNWSLTSPGTTSAILADVAASDEDRSGGIIHNQ FVQIFFYVLYATVFVLGVFGNVLVCYVVLRNRAMQTVTNIFITNLALSDILLCVLAVPFT PLYTFMGRWAFGRSLCHLVSFAQGCSIYISTLTLTSIAIDRYFVIIYPFHPRMKLSTCIG IIVSIWVIALLATVPYGMYMKMTNELVNGTQTGNETLVEATLMLNGSFVAQGSGFIEAPD STSATQAYMQVMTAGSTGPEMPYVRVYCEENWPSEQYRKVFGAITTTLQFVLPFFIISIC YVWISVKLNQRARAKPGSKSSRREEADRDRKKRTNRMLIAMVAVFGLSWLPINVVNIFDD FDDKSNEWRFYILFFFVAHSIAMSSTCYNPFLYAWLNENFRKEFKHVLPCFNPSNNNIIN ITRGYNRSDRNTCGPRLHHGKGDGGMGGGSLDADDQDENGITQETCLPKEKLLIIPREPT YGNGTGAVSPILSGRGINAALVHGGDHQMHQLQPSHHQQVELTRRIRRRTDETDGDYLDS GDEQTVEVRFSETPFVSTDNTTGISILETSTSHCQDSDVMVELGEAIGAGGGAELGRRIN

DmGPCR2a ( SEQ ID NO：3 ) DNA： ATGAATCAGACGGAGCCCGCCCAGCTGGCAGATGGGGAGCATCTGAGTGG ATACGCCAGCAGCAGCAACAGCGTGCGCTATCTGGACGACCGGCATCCGC TGGACTACCTTGACCTGGGCACGGTGCACGCCCTCAACACCACTGCCATC AACACCTCGGATCTGAATGAGACTGGGAGCAGGCCGCTGGACCCGGTGCT TATCGATAGGTTCCTGAGCAACAGGGCGGTGGACAGCCCCTGGTACCACA TGCTCATCAGCATGTACGGCGTGCTAATCGTCTTCGGCGCCCTAGGCAAC ACCCTGGTTGTTATAGCCGTCATCCGGAAGCCCATCATGCGCACTGCTCG CAATCTGTTCATCCTCAACCTGGCCATATCGGACCTACTTTTATGCCTAG TCACCATGCCGCTGACCTTGATGGAGATCCTGTCCAAGTACTGGCCCTAC GGCTCCTGCTCCATCCTGTGCAAAACGATTGCCATGCTGCAGGCACTTTG TATTTTCGTGTCGACAATATCCATAACGGCCATTGCCTTCGACAGATATC AGGTGATCGTGTACCCCACGCGGGACAGCCTGCAGTTCGTGGGCGCGGTG ACGATCCTGGCGGGGATCTGGGCACTGGCACTGCTGCTGGCCTCGCCGCT GTTCGTCTACAAGGAGCTGATCAACACAGACACGCCGGCACTCCTGCAGC AGATCGGCCTGCAGGACACGATCCCGTACTGCATTGAGGACTGGCCAAGT CGCAACGGGCGCTTCTACTACTCGATCTTCTCGCTGTGCGTACAATACCT GGTGCCCATCCTGATCGTCTCGGTGGCATACTTCGGGATCTACAACAAGC TGAAGAGCCGCATCACCGTGGTGGCTGTGCAGGCGTCCTCCGCTCAGCGG AAGGTGGAGCGGGGGCGGCGGATGAAGCGCACCAACTGCCTACTGATCAG CATCGCCATCATCTTTGGCGTTTCTTGGCTGCCGCTGAACTTTTTCAACC TGTACGCGGACATGGAGCGCTCGCCGGTCACTCAGAGCATGCTAGTCCGC TACGCCATCTGCCACATGATCGGCATGAGCTCCGCCTGCTCCAACCCGTT GCTCTACGGCTGGCTCAACGACAACTTCCGTAAAGAATTTCAAGAACTGC TCTGCCGTTGCTCAGACACTAATGTTGCTCTTAACGGTCACACGACAGGC TGCAACGTCCAGGCGGCGGCGCGCAAGCGTCGCAAGTTGGGCGCCGAACT CTCCAAAGGCGAACTCAAGCTGCTGGGGCCAGGCGGCGCCCAGAGCGGTA CCGCCGGCGGGGAAGGCGGTCTGGCGGCCACCGACTTCATGACCGGCCAC CACGAGGGCGGACTGCGCAGCGCCATAACCGAGTCGGTGGCCCTCACGGA CCACAACCCCGTGCCCTCGGAGGTCACCAAGCTGATGCCGCGGTA ( SEQ ID NO：4 ) SEQ ID NO：3DNA ： MENTTMLANISLNATRNEENITSFFTDEEWLAINGTLPWIVGFFFGVIAITGFFGNLLVILVVVFN NNMRSTTNLMIVNLAAADLMFVILCIPFTATDYMVYYWPYGRFWCRSVQYLIVVTAFASIYTLVLM SIDRFLAVVHPIRSRMMRTENITLIAIVTLWIVVLVVSVPVAFTHDVVVDYDAKKNITYGMCTFTT NDFLGPRTYQVTFFISSYLLPLMIISGLYMRMIMRLWRQGTGVRMSKESQRGRKRVTRLVVVVVIA FASLWLPVQLILLLKSLDVIETNTLTKLVIQVTAQTLAYSSSCINPLLYAFLSENFRKAFYKAVNC SSRYQNYTSDLPPPRKTSCARTSTTGL

DmGPCR2b ( SEQ ID NO：5 ) DNA： ATGAATCAGACGGAGCCCGCCCAGCTGGCAGATGGGGAGCATCTGAGTGG ATACGCCAGCAGCAGCAACAGCGTGCGCTATCTGGACGACCGGCATCCGC TGGACTACCTTGACCTGGGCACGGTGCACGCCCTCAACACCACTGCCATC AACACCTCGGATCTGAATGAGACTGGGAGCAGGCCGCTGGACCCGGTGCT TATCGATAGGTTCCTGAGCAACAGGGCGGTGGACAGCCCCTGGTACCACA TGCTCATCAGCATGTACGGCGTGCTAATCGTCTTCGGCGCCCTAGGCAAC ACCCTGGTTGTTATAGCCGTCATCCGGAAGCCCATCATGCGCACTGCTCG CAATCTGTTCATCCTCAACCTGGCCATATCGGACCTACTTTTATGCCTAG TCACCATGCCGCTGACCTTGATGGAGATCCTGTCCAAGTACTGGCCCTAC GGCTCCTGCTCCATCCTGTGCAAAACGATTGCCATGCTGCAGGCACTTTG TATTTTCGTGTCGACAATATCCATAACGGCCATTGCCTTCGACAGATATC AGGTGATCGTGTACCCCACGCGGGACAGCCTGCAGTTCGTGGGCGCGGTG ACGATCCTGGCGGGGATCTGGGCACTGGCACTGCTGCTGGCCTCGCCGCT GTTCGTCTACAAGGAGCTGATCAACACAGACACGCCGGCACTCCTGCAGC AGATCGGCCTGCAGGACACGATCCCGTACTGCATTGAGGACTGGCCAAGT CGCAACGGGCGCTTCTACTACTCGATCTTCTCGCTGTGCGTACAATACCT GGTGCCCATCCTGATCGTCTCGGTGGCATACTTCGGGATCTACAACAAGC TGAAGAGCCGCATCACCGTGGTGGCTGTGCAGGCGTCCTCCGCTCAGCGG AAGGTGGAGCGGGGGCGGCGGATGAAGCGCACCAACTGCCTACTGATCAG CATCGCCATCATCTTTGGCGTTTCTTGGCTGCCGCTGAACTTTTTCAACC TGTACGCGGACATGGAGCGCTCGCCGGTCACTCAGAGCATGCTAGTCCGC TACGCCATCTGCCACATGATCGGCATGAGCTCCGCCTGCTCCAACCCGTT GCTCTACGGCTGGCTCAACGACAACTTCCGCTGCAACGTCCAGGCGGCGG CGCGCAAGCGTCGCAAGTTGGGCGCCGAACTCTCCAAAGGCGAACTCAAG CTGCTGGGGCCAGGCGGCGCCCAGAGCGGTACCGCCGGCGGGGAAGGCGG TCTGGCGGCCACCGACTTCATGACCGGCCACCACGAGGGCGGACTGCGCA GCGCCATAACCGAGTCGGTGGCCCTCACGGACCACAACCCCGTGCCCTCG GAGGTCACCAAGCTGATGCCGCGGTA ( SEQ ID NO：6 ) SEQ ID NO：5DNA ： MNQTEPAQLADGEHLSGYASSSNSVRYLDDRHPLDYLDLGTVHALNTTAINTSDLNETGSRPLDPV LIDRFLSNRAVDSPWYHMLISMYGVLIVFGALGNTLVVIAVIRKPIMRTARNLFILNLAISDLLLC LVTMPLTLMEILSKYWPYGSCSILCKTIAMLQALCIFVSTISITAIAFDRYQVIVYPTRDSLQFVG AVTILAGIWALALLLASPLFVYKELINTDTPALLQQIGLQDTIPYCIEDWPSRNGRFYYSIFSLCV QYLVPILIVSVAYFGIYNKLKSRITVVAVQASSAQRKVERGRRMKRTNCLLISIAIIFGVSWLPLN FFNLYADMERSPVTQSMLVRYAICHMIGMSSACSNPLLYGWLNDNFRCNVQAAARKRRKLGAELSK GELKLLGPGGAQSGTAGGEGGLAATDFMTGHHEGGLRSAITESVALTDHNPVPSEVTKLMPR

DmGPCR4 ( SEQ ID NO：7 ) DNA： ATGGAGAACACCACAATGCTGGCTAATATTAGCCTAAATGCAACCAGAAA TGAGGAGAATATCACCTCATTCTTCACCGACGAAGAGTGGCTGGCCATCA ATGGCACTTTGCCGTGGATAGTGGGATTCTTCTTCGGCGTCATCGCCATC ACGGGATTCTTCGGCAACCTGCTGGTCATCCTGGTGGTGGTCTTCAACAA CAACATGCGCTCCACCACCAACCTGATGATTGTCAATCTGGCTGCCGCTG ATCTGATGTTCGTAATCCTCTGCATTCCCTTCACGGCCACCGATTACATG GTGTACTACTGGCCATATGGAAGGTTCTGGTGCCGCAGTGTCCAGTACCT GATTGTGGTGACCGCCTTCGCCTCCATCTACACGCTGGTGCTAATGTCCA TCGATCGGTTCCTGGCGGTGGTTCATCCCATTCGCTCGCGGATGATGAGG ACGGAGAACATTACCCTGATTGCCATCGTGACTCTGTGGATCGTGGTGCT GGTCGTTTCGGTGCCAGTGGCCTTCACCCACGACGTGGTGGTGGACTACG ATGCAAAGAAGAACATCACCTACGGCATGTGCACCTTCACGACGAACGAC TTCCTTGGTCCGCGCACCTACCAGGTCACCTTCTTCATCAGCTCCTACCT GCTGCCCCTGATGATCATCAGCGGTCTCTACATGCGCATGATCATGCGGC TCTGGCGCCAGGGAACCGGCGTCCGCATGTCCAAGGAGTCGCAGCGCGGT CGCAAGCGGGTCACCCGACTCGTCGTCGTGGTGGTCATCGCCTTCGCCTC GCTCTGGCTGCCTGTCCAGCTCATCCTGCTGCTCAAGTCACTGGATGTCA TCGAGACGAACACCCTCACCAAGCTAGTCATCCAGGTCACCGCCCAGACT CTGGCCTACAGCAGCTCGTGTATCAATCCGCTGCTCTACGCCTTCCTCTC CGAGAATTTCCGGAAGGCCTTCTATAAGGCCGTTAACTGCTCCTCTCGAT

ACCAGAACTACACATCTGATTTGCCGCCGCCGCGCAAGACGTCCTGTGCC AGGACCTCCACCACTGGACTCTA following amino acid sequences (SEQ ID NO:8) is the amino acid sequence of protein of the dna sequence encoding of SEQ ID NO:7: MENTTMLANISLNATRNEENITSFFTDEEWLAINGTLPWIVGFFFGVIAITGFFGN LLVILVVVFN NNMRSTTNLMIVNLAAADLMFVILCIPFTATDYMVYYWPYGRFWCRSVQYLIVVTA FASIYTLVLM SIDRFLAVVHPIRSRMMRTENITLIAIVTLWIVVLVVSVPVAFTHDVVVDYDAKKN ITYGMCTFTT NDFLGPRTYQVTFFISSYLLPLMIISGLYMRMIMRLWRQGTGVRMSKESQRGRKRV TRLVVVVVIA FASLWLPVQLILLLKSLDVIETNTLTKLVIQVTAQTLAYSSSCINPLLYAFLSENF RKAFYKAVNC SSRYQNYTSDLPPPRKTSCARTSTTGL

DmGPCR5 ( SEQ ID NO：9 ) DNA： ATGGAGAATCGCAGTGACTTCGAGGCGGATGACTACGGCGACATCAGTTG GAGCAATTGGAGCAACTGGAGCACCCCCGCCGGCGTCCTTTTCTCGGCCA TGAGCAGCGTGCTCTCGGCCAGCAACCATACGCCCCTGCCGGACTTTGGC CAGGAGCTCGCCCTATCCACCAGCTCCTTCAATCACAGCCAGACCCTATC CACCGACCAGCCCGCCGTCGGGGACGTGGAAGACGCGGCCGAGGATGCGG CGGCGTCCATGGAGACGGGCTCGTTTGCATTTGTGGTCCCGTGGTGGCGT CAGGTGCTCTGGAGCATCCTCTTCGGCGGCATGGTCATTGTGGCGACGGG CGGTAACCTGATTGTTGTCTGGATCGTGATGACGACCAAGCGGATGCGGA CGGTAACCAACTATTTCATAGTGAATCTCTCCATCGCGGACGCCATGGTG TCCAGCCTAAACGTCACCTTCAACTACTACTATATGCTGGATAGCGACTG GCCCTTCGGCGAGTTCTACTGCAAGTTGTCCCAGTTCATCGCGATGCTAA GCATCTGCGCCTCAGTGTTCACCCTAATGGCCATCTCCATCGACAGATAC GTGGCCATCATCCGGCCACTGCAGCCGCGGATGAGCAAGCGGTGCAACCT GGCCATCGCGGCGGTCATCTGGCTGGCCTCCACGCTCATCTCCTGCCCCA TGATGATCATCTACCGCACGGAGGAGGTGCCGGTCCGCGGGCTCAGCAAC CGCACGGTCTGCTACCCGGAGTGGCCCGATGGGCCCACCAATCACTCCAC GATGGAGTCCCTCTACAACATCCTCATCATCATYCTAACCTACTTCCTGC CCATCGTCTCCATGACGGTCACCTACTCGCGCGTGGGCATCGAGCTCTGG GGATCCAAGACCATCGGCGAGTGCACGCCCCGCCAGGTGGARAAYGTGCG GAGTAAGCGAAGGGTGGTGAAGATGATGATTGTGGTCGTCCTGATATTCG CCATCTGCTGGCTGCCGTTCCACAGCTACTTCATAATCACATCCTGCTAC CCGGCCATCACGGAGGCGCCCTTCATCCAGGAACTCTACCTGGCCATCTA CTGGCTGGCCATGAGCAACTCCATGTACAATCCCATTATATACTGCTGGA TGAATTCGCGCTTTCGCTATGGTTTCAAGATGGTCTTCCGCTGGTGCCTG TTTGTGCGCGTGGGCACTGAACCCTTTAGTCGGCGGGAGAACCTGACATC CCGGTACTCCTGCTCCGGTTCCCCGGATCACAATCGCATCAAGCGCAATG ATACCCAGAAATCGATACTTTATACCTGTCCCAGCTCACCCAAGTCGCAT CGAATTTCGCACAGCGGAACAGGTCGCAGTGCGACGCTGCGGAACAGTCT GCCGGCGGAGTCACTGTCGTCCGGCGGATCTGGTGGTGGAGGGCACAGGA AACGGTTGTCCTACCAGCAGGAAATGCAGCAGCGTTGGTCAGGACCCAAT AGTGCCACCGCAGTGACCAATTCCAGCAGTACGGCCAACACCACCCAACT GCTCTCCTG ( SEQ ID NO：10 ) SEQ ID NO：9DNA ： MENRSDFEADDYGDISWSNWSNWSTPAGVLFSAMSSVLSASNHTPLPDFGQELALSTSSFNHSQTL STDQPAVGDVEDAAEDAAASMETGSFAFVVPWWRQVLWSILFGGMVIVATGGNLIVVWIVMTTKRM RTVTNYFIVNLSIADAMVSSLNVTFNYYYMLDSDWPFGEFYCKLSQFIAMLSICASVFTLMAISID RYVAIIRPLQPRMSKRCNLAIAAVIWLASTLISCPMMIIYRTEEVPVRGLSNRTVCYPEWPDGPTN HSTMESLYNILIIILTYFLPIVSMTVTYSRVGIELWGSKTIGECTPRQVENVRSKRRVVKMMIVVV LIFAICWLPFHSYFIITSCYPAITEAPFIQELYLAIYWLAMSNSMYNPIIYCWMNSRFRYGFKMVF RWCLFVRVGTEPFSRRENLTSRYSCSGSPDHNRIKRNDTQKSILYTCPSSPKSHRISHSGTGRSAT LRNSLPAESLSSGGSGGGGHRKRLSYQQEMQQRWSGPNSATAVTNSSSTANTTQLLS DmGPCR5b ( SEQ ID NO：11 ) DNA： ATGGAGAATCGCAGTGACTTCGAGGCGGATGACTACGGCGACATCAGTTG GAGCAATTGGAGCAATTGGAGCAACTGGAGCACCCCCGCCGGCGTCCTTT TCTCGGCCATGAGCAGCGTGCTCTCGGCCAGCAACCATACGCCTCTGCCG GACTTTGGCCAGGAGCTCGCCCTATCCACCAGCTCCTTCAATCACAGCCA

GACCCTATCCACCGACCTGCCCGCCGTCGGGGACGTGGAAGACGCGGCCG AGGATGCGGCGGCGTCCATGGAGACGGGCTCGTTTGCATTTGTGGTCCCG TGGTGGCGTCAGGTGCTCTGGAGCATCCTCTTCGGCGGCATGGTCATTGT GGCGACGGGCGGTAACCTGATTGTTGTCTGGATCGTGATGACGACCAAGC GGATGCGGACGGTAACCAACTATTTCATAGTAAATCTCTCCATCGCGGAC GCCATGGTGTCCAGCCTGAACGTCACCTTCAACTACTACTACATGCTGGA TAGCGACTGGCCCTTCGGCGAGTTCTACTGCAAGTTGTCCCAGTTCATCG CGATGCTAAGCATCTGCGCCTCAGTGTTCACCCTAATGGCCATCTCCATC GACAGATACGTGGCCATCATCCGGCCACTGCAGCCGCGGATGAGCAAGCG GTGCAACCTGGCCATCGCGGCGGTCATCTGGCTGGCCTCCACGCTCATCT CCTGCCCCATGATGATCATCTACCGCACGGAGGAGGTGCCGGTCCGCGGG CTCAGCAACCGCACGGTCTGCTACCCGGAGTGGCCCGATGGGCCCACCAA TCACTCCACGATGGAGTCCCTCTACAACATCCTCATCATCATTCTAACCT ACTTCCTGCCCATCGTCTCCATGACGGTCACCTACTCGCGCGTGGGCATC GAGCTCTGGGGATCCAAGACCATCGGCGAGTGCACGCCCCGCCAGGTGGA GAATGTGCGGAGTAAGCGAAGGGTGGTGAAGATGATGATTGTGGTCGTCC TGATATTCGCCATCTGCTGGCTGCCGTTCCACAGCTACTTCATAATCACA TCCTGCTACCCGGCCATCACGGAGGCGCCCTTCATCCAGGAACTTTACCT GGCCATCTACTGGCTGGCCATGAGCAACTCCATGTACAATCCCATTATAT ACTGCTGGATGAATTCGCGCTTTCGCTATGGTTTCAAGATGGTCTTCCGC TGGTGCCTGTTTGTGCGCGTGGGCACTGAACCCTTTAGTCGGCGGGAGAA CCTGACATCCCGGTACTCCTGCTCCGGTTCCCCGGATCACAATCGCATCA AGCGCAATGATACCCAGAAATCGATACTTTATACCTGTCCCAGCTCACCC AAGTCGCATCGAATTTCGCACAGCGGAACAGGTCGCAGTGCGACGCTGAG GAACAGTCTGCCGGCGGAGTCATTGTCGTCCGGTGGATCTGGAGGTGGAG GACACAGGAAACGGTTGTCCTACCAGCAGGAAATGCAGCAGCGGTGGTCA GGACCCAATAGTGCCACCGCAGTGACCAATTCCAGCAGTACGGCCAACAC CACCCAACTGCTCTCCTG ( SEQ ID NO：12 ) SEQ ID NO：11DNA ： MENRSDFEADDYGDISWSNWSNWSNWSTPAGVLFSAMSSVLSASNHTPLPDFGQELALSTSSFNHS QTLSTDLPAVGDVEDAAEDAAASMETGSFAFVVPWWRQVLWSILFGGMVIVATGGNLIVVWIVMTT KRMRTVTNYFIVNLSIADAMVSSLNVTFNYYYMLDSDWPFGEFYCKLSQFIAMLSICASVFTLMAI SIDRYVAIIRPLQPRMSKRCNLAIAAVIWLASTLISCPMMIIYRTEEVPVRGLSNRTVCYPEWPDG PTNHSTMESLYNILIIILTYFLPIVSMTVTYSRVGIELWGSKTIGECTPRQVENVRSKRRVVKMMI VVVLIFAICWLPFHSYFIITSCYPAITEAPFIQELYLAIYWLAMSNSMYNPIIYCWMNSRFRYGFK MVFRWCLFVRVGTEPFSRRENLTSRYSCSGSPDHNRIKRNDTQKSILYTCPSSPKSHRISHSGTGR SATLRNSLPAESLSSGGSGGGGHRKRLSYQQEMQQRWSGPNSATAVTNSSSTANTTQLLS

DmGPCR6aL ( SEQ ID NO：13 ) DNA ATGGAGCACCACAATAGCCATCTGTTGCCTGGTGGCAGCGAGAAGATGTA CTACATAGCTCACCAGCAGCCGATGCTGCGGAACGAGGATGATAACTACC AGGAGGGGTACTTCATCAGGCCGGACCCTGCATCCTTACTTTACAATACC ACCGCACTGCCAGCGGACGATGAAGGGTCCAACTATGGATATGGCTCCAC CACAACGCTCAGTGGCCTCCAGTTCGAGACCTATAATATCACTGTGATGA TGAACTTTAGCTGTGACGACTATGACCTTCTATCGGAGGACATGTGGTCT AGTGCCTACTTTAAGATCATCGTCTACATGCTCTACATTCCCATCTTTAT CTTCGCCCTGATCGGCAACGGAACGGTCTGCTATATCGTCTATTCCACAC CTCGCATGCGCACGGTCACCAATTACTTTATAGCCAGCTTGGCCATCGGC GACATCCTGATGTCCTTCTTCTGCGTTCCGTCGTCCTTCATCTCGCTGTT CATCCTGAACTACTGGCCTTTTGGCCTGGCCCTCTGTCACTTTGTGAACT ACTCGCAGGCGGTCTCAGTTCTGGTCAGCGCCTATACTTTGGTGGCAATT AGCATTGACCGCTACATAGCCATTATGTGGCCATTAAAGCCACGCATCAC AAAACGCTATGCCACCTTCATCATCGCCGGCGTTTGGTTTATTGCACTTG CCACCGCACTTCCCATACCCATCGTCTCTGGACTCGACATCCCAATGTCG CCGTGGCACACGAAATGCGAGAAATACATTTGCCGCGAAATGTGGCCGTC GCGGACGCAGGAGTACTACTACACCCTGTCCCTCTTCGCGCTGCAGTTCG TCGTGCCGCTGGGCGTGCTCATCTTCACCTACGCCCGGATCACCATTCGC GTCTGGGCGAAACGACCGCCAGGCGAGGCGGAAACCAACCGCGACCAGCG GATGGCACGCTCCAAACGGAAGATGGTCAAAATGATGCTGACGGTTGTGA TTGTGTTCACCTGCTGTTGGCTGCCCTTCAATATTTTGCAGCTTTTACTG

AACGACGAGGAGTTCGCCCACTGGGATCCTCTGCCGTATGTATGGTTCGC GTTTCACTGGCTGGCCATGTCGCACTGCTGCTACAATCCGATCATCTACT GCTACATGAACGCCCGTTTCAGGAGCGGATTCGTCCAGCTGATGCACCGT ATGCCCGGCCTGCGTCGCTGGTGCTGCCTGCGGAGCGTCGGTGATCGCAT GAACGCAACTTCCGGAACGGGTCCAGCACTTCCTCTCAATCGAATGAACA CATCCACCACCTACATCAGCGCTCGTCGAAAGCCACGAGCGACATCTTTG CGAGCGAACCCATTATCATGCGGCGAGACGTCACCACTGCGGTA ( SEQ ID NO：14 ) SEQ ID NO：13DNA ： MEHHNSHLLPGGSEKMYYIAHQQPMLRNEDDNYQEGYFIRPDPASLLYNTTALPADDEGSNYGYGS TTTLSGLQFETYNITVMMNFSCDDYDLLSEDMWSSAYFKIIVYMLYIPIFIFALIGNGTVCYIVYS TPRMRTVTNYFIASLAIGDILMSFFCVPSSFISLFILNYWPFGLALCHFVNYSQAVSVLVSAYTLV AISIDRYIAIMWPLKPRITKRYATFIIAGVWFIALATALPIPIVSGLDIPMSPWHTKCEKYICREM WPSRTQEYYYTLSLFALQFVVPLGVLIFTYARITIRVWAKRPPGEAETNRDQRMARSKRKMVKMML TVVIVFTCCWLPFNILQLLLNDEEFAHWDPLPYVWFAFHWLAMSHCCYNPIIYCYMNARFRSGFVQ LMHRMPGLRRWCCLRSVGDRMNATSGTGPALPLNRMNTSTTYISARRKPRATSLRANPLSCGETSP LR DmGPCR6bL ( SEQ ID NO：15 ) DNA： ATGGAGCACCACAATAGCCATCTGTTGCCTGGTGGCAGCGAGAAGATGTA CTACATAGCTCACCAGCAGCCGATGCTGCGGAACGAGGATGATAACTACC AGGAGGGGTACTTCATCAGGCCGGACCCTGCATCCTTACTTTACAATACC ACCGCACTGCCAGCGGACGATGAAGGGTCCAACTATGGATATGGCTCCAC CACAACGCTCAGTGGCCTCCAGTTCGAGACCTATAATATCACTGTGATGA TGAACTTTAGCTGTGACGACTATGACCTTCTATCGGAGGACATGTGGTCT AGTGCCTACTTTAAGATCATCGTCTACATGCTCTACATTCCCATCTTTAT CTTCGCCCTGATCGGCAACGGAACGGTCTGCTATATCGTCTATTCCACAC CTCGCATGCGCACGGTCACCAATTACTTTATAGCCAGCTTGGCCATCGGC GACATCCTGATGTCCTTCTTCTGCGTTCCGTCGTCCTTCATCTCGCTGTT CATCCTGAACTACTGGCCTTTTGGCCTGGCCCTCTGTCACTTTGTGAACT ACTCGCAGGCGGTCTCAGTTCTGGTCAGCGCCTATACTTTGGTGGCAATT AGCATTGACCGCTACATAGCCATTATGTGGCCATTAAAGCCACGCATCAC AAAACGCTATGCCACCTTCATCATCGCCGGCGTTTGGTTTATTGCACTTG CCACCGCACTTCCCATACCCATCGTCTCTGGACTCGACATCCCAATGTCG CCGTGGCACACGAAATGCGAGAAATACATTTGCCGCGAAATGTGGCCGTC GCGGACGCAGGAGTACTACTACACCCTGTCCCTCTTCGCGCTGCAGTTCG TCGTGCCGCTGGGCGTGCTCATCTTCACCTACGCCCGGATCACCATTCGC GTCTGGGCGAAACGACCGCCAGGCGAGGCGGAAACCAACCGCGACCAGCG GATGGCACGCTCCAAACGGAAGATGGTCAAAATGATGCTGACGGTTGTGA TTGTGTTCACCTGCTGTTGGCTGCCCTTCAATATTTTGCAGCTTTTACTG AACGACGAGGAGTTCGCCCACTGGGATCCTCTGCCGTATGTGTGGTTCGC GTTTCACTGGCTGGCCATGTCGCACTGCTGCTACAATCCGATCATCTACT GCTACATGAACGCCCGTTTCAGGAGCGGATTCGTCCAGCTGATGCACCGT ATGCCCGGCCTGCGTCGCTGGTGCTGCCTGCGGAGCGTCGGTGATCGCAT GAACGCAACTTCCGGTGAGATGACTACGAAGTACCATCGCCATGTCGGCG ATGCCCTATTCCGGAAACCCAAAATATGCATTAGGAACGGGTCCAGCACT TCCTCTCAATCGAATGAACACATCCACCACCTACATCAGCGCTCGTCGAA AGCCACGAGCGACATCTTTGCGAGCGAACCCATTATCATGCGGCGAGACG TCACCACTGCGGTAGCTGTCATATCAAAAAATAAAACTGATTCACCGGTG CGCCGATCGGGAAGCTCAGGTGGAACAGAAGCAAACATAAGAAGCACCGA GTTTTG ( SEQ ID NO：16 ) SEQ ID NO：15DNA ： MEHHNSHLLPGGSEKMYYIAHQQPMLRNEDDNYQEGYFIRPDPASLLYNTTALPADDEGSNYGYGS TTTLSGLQFETYNITVMMNFSCDDYDLLSEDMWSSAYFKIIVYMLYIPIFIFALIGNGTVCYIVYS TPRMRTVTNYFIASLAIGDILMSFFCVPSSFISLFILNYWPFGLALCHFVNYSQAVSVLVSAYTLV AISIDRYIAIMWPLKPRITKRYATFIIAGVWFIALATALPIPIVSGLDIPMSPWHTKCEKYICREM WPSRTQEYYYTLSLFALQFVVPLGVLIFTYARITIRVWAKRPPGEAETNRDQRMARSKRKMVKMML TVVIVFTCCWLPFNILQLLLNDEEFAHWDPLPYVWFAFHWLAMSHCCYNPIIYCYMNARFRSGFVQ LMHRMPGLRRWCCLRSVGDRMNATSGEMTTKYHRHVGDALFRKPKICIRNGSSTSSQSNEHIHHLH

QRSSKATSDIFASEPIIMRRDVTTAVAVISKNKTDSPVRRSGSSGGTEANIRSTEF
	DmGPCR7 ( SEQ ID NO：17 ) DNA： ATGGCAATGGACTTAATCGAGCAGGAGTCCCGCCTGGAATTCCTGCCCGG AGCCGAGGAGGAAGCAGAATTTGAGCGTCTATACGCGGCTCCCGCTGAGA TTGTGGCCCTGTTGTCCATTTTCTATGGGGGAATCAGTATCGTGGCCGTC ATTGGCAACACTTTGGTCATCTGGGTGGTGGCCACGACCAGGCAAATGCG GACCGTGACAAATATGTATATCGCTAATTTGGCTTTTGCCGATGTGATTA TTGGCCTCTTCTGCATACCATTTCAGTTCCAGGCTGCCCTGCTGCAGAGT TGGAACCTGCCGTGGTTCATGTGCAGCTTCTGCCCCTTCGTCCAGGCCCT GAGTGTAAATGTCTCGGTATTCACGCTGACCGCCATTGCAATCGATCGGC ATAGGGCCATCATTAATCCACTTAGGGCACGTCCCACCAAGTTCGTATCG AAGTTCATAATTGGTGGAATTTGGATGCTGGCCCTGCTATTTGCGGTGCC CTTTGCCATTGCCTTTCGTGTGGAGGAGTTGACCGAAAGATTTCGCGAGA ACAATGAGACCTACAATGTGACGCGGCCATTCTGCATGAACAAGAACCTA TCCGATGATCAATTGCAATCCTTTCGCTACACCCTGGTTTTTGTGCAGTA TCTGGTTCCATTCTGTGTCATCAGCTTTGTCTACATCCAGATGGCGGTAC GATTGTGGGGCACACGTGCTCCTGGTAACGCACAGGATTCACGGGACATA ACGCTGTTGAAAAACAAGAAGAAGGTCATCAAAATGCTGATTATCGTGGT CATTATCTTTGGACTCTGCTGGCTGCCACTGCAGCTCTATAATATTCTGT ATGTCACGATACCGGAAATCAACGACTACCACTTCATTAGCATCGTCTGG TTTTGCTGCGATTGGCTGGCCATGAGCAATAGCTGCTACAATCCCTTTAT TTATGGCATCTACAATGAAAAATTTAAGCGGGAATTCAACAAGCGATTTG CGGCCTGTTTCTGCAAGTTCAAGACGAGCATGGACGCCCACGAAAGGACC TTTTCGATGCACACCCGCGCCAGCTCCATAAGGTCAACCTACGCCAACTC CTCGATGCGAATCCGGAGTAATCTCTTTGGTCCGGCGCGTGGTGGTGTCA ACAATGGGAAGCCGGGCTTGCATATGCCGCGGGTGCATGGATCCGGTGCT AACAGCGGCATTTACAACGGAAGTAGTGGGCAGAACAACAATGTCAATGG CCAACATCATCAGCATCAAAGCGTGGTTACCTTTGCGGCCACTCCGGGTG TTTCGGCACCAGGTGTTGGCGTTGCAATGCCGCCGTGGCGGCGAAACAAC TTCAAACCTCTGCATCCGAACGTAATCGAATGCGAGGACGACGTGGCACT CATGGAGCTGCCATCAACCACGCCCCCCAGCGAGGAGTTGGCATCCGGGG CCGGAGTCCAGTTGGCCCTGCTAAGCAGGGAGAGCTCCAGCTGCATTTGC GAACAGGAATTTGGCAGCCAAACCGAATGCGATGGCACCTGCATACTCAG CGAGGTGTCGCGAGTCCACCTGCCCGGCTCGCAGGCGAAGGACAAGGATG CGGGCAAGTCCTTGTGGCAACCACTTTA ( SEQ ID NO：18 ) SEQ ID NO：17DNA ： MAMDLIEQESRLEFLPGAEEEAEFERLYAAPAEIVALLSIFYGGISIVAVIGNTLVIWVVATTRQM RTVTNMYIANLAFADVIIGLFCIPFQFQAALLQSWNLPWFMCSFCPFVQALSVNVSVFTLTAIAID RHRAIINPLRARPTKFVSKFIIGGIWMLALLFAVPFAIAFRVEELTERFRENNETYNVTRPFCMNK NLSDDQLQSFRYTLVFVQYLVPFCVISFVYIQMAVRLWGTRAPGNAQDSRDITLLKNKKKVIKMLI IVVIIFGLCWLPLQLYNILYVTIPEINDYHFISIVWFCCDWLAMSNSCYNPFIYGIYNEKFKREFN KRFAACFCKFKTSMDAHERTFSMHTRASSIRSTYANSSMRIRSNLFGPARGGVNNGKPGLHMPRVH GSGANSGIYNGSSGQNNNVNGQHHQHQSVVTFAATPGVSAPGVGVAMPPWRRNNFKPLHPNVIECE DDVALMELPSTTPPSEELASGAGVQLALLSRESSSCICEQEFGSQTECDGTCILSEVSRVHLPGSQ AKDKDAGKSLWQPL
DmGPCR8 ( SEQ ID NO：19 ) DNA ATGTTTACGTGGCTGATGATGGATGTCCTCCAGTTTGTGAAAGGGGAAAT GACAGCCGATTCAGAGGCAAATGCCACAAATTGGTATAACACGAACGAGA GCTTATATACCACGGAACTGAACCATAGATGGATTAGTGGTAGTTCCACA ATTCAGCCAGAGGAGTCCCTTTATGGCACTGATTTGCCCACCTATCAACA TTGCATAGCCACGCGGAATTCCTTTGCTGACTTGTTCACTGTGGTGCTCT ACGGATTTGTGTGCATTATCGGATTATTTGGCAACACCCTGGTGATCTAC GTGGTGTTGCGCTTTTCCAAAATGCAAACGGTCACGAATATATATATCCT GAATCTGGCGGTGGCAGACGAGTGCTTCCTGATTGGAATACCCTTTCTGC TGTACACAATGCGAATTTGCAGCTGGCGATTCGGGGAGTTTATGTGCAAA GCCTACATGGTGAGCACATCCATCACCTCCTTCACCTCGTCGATTTTTCT GCTCATCATGTCCGCGGATCGATATATAGCGGTATGCCACCCGATTTCCT

CGCCACGATATCGAACTCTGCATATTGCCAAAGTGGTCTCAGCGATTGCC TGGTCAACTTCAGCGGTCCTCATGCTGCCCGTGATCCTTTATGCCAGCAC TGTGGAGCAGGAGGATGGCATCAATTACTCGTGCAACATAATGTGGCCAG ATGCGTACAAGAAGCATTCGGGCACCACCTTCATACTGTACACATTTTTC CTAGGATTCGCCACACCGCTGTGCTTTATCCTGAGTTTCTACTACTTGGT TATAAGGAAACTGCGATCGGTGGGTCCCAAACCAGGAACGAAGTCCAAGG AGAAGAGGCGGGCTCACAGGAAGGTCACTCGACTGGTACTGACGGTGATA AGTGTATACATTCTATGTTGGCTCCCTCACTGGATTTCTCAGGTGGCCCT GATTCACTCGAATCCCGCGCAAAGGGACCTCTCCCGACTGGAAATACTCA TTTTCCTACTTCTGGGGGCACTGGTTTACTCGAATTCGGCGGTGAATCCC ATACTTTATGCCTTCCTAAGTGAGAACTTCCGGAAGAGCTTCTTCAAGGC CTTTACCTGTATGAATAAGCAGGATATCAACGCTCAACTCCAGCTGGAGC CCAGTGTTTTCACCAAACAGGGCAGTAAAAAGAGGGGTGGCTCCAAGCGC CTGTTGACCAGCAATCCGCAGATTCCTCCACTGCTGCCACTGAATGCGGG TAACAACAATTCATCGACCACCACATCCTCGACCACGACAGCGGAAAAGA CCGGAACCACGGGGACACAGAAATCATGCAATTCCAATGGCAAAGTGACA GCTCCGCCGGAGAATTTGATTATATGTTTGAGCGAGCAGCAGGAGGCATT TTGCACCACCGCGAGAAGAGGATCGGGCGCAGTGCAGCAGACAGATTTGT A ( SEQ ID NO：20 ) SEQ ID NO：19DNA ： MFTWLMMDVLQFVKGEMTADSEANATNWYNTNESLYTTELNHRWISGSSTIQPEESLYGTDLPTYQ HCIATRNSFADLFTVVLYGFVCIIGLFGNTLVIYVVLRFSKMQTVTNIYILNLAVADECFLIGIPF LLYTMRICSWRFGEFMCKAYMVSTSITSFTSSIFLLIMSADRYIAVCHPISSPRYRTLHIAKVVSA IAWSTSAVLMLPVILYASTVEQEDGINYSCNIMWPDAYKKHSGTTFILYTFFLGFATPLCFILSFY YLVIRKLRSVGPKPGTKSKEKRRAHRKVTRLVLTVISVYILCWLPHWISQVALIHSNPAQRDLSRL EILIFLLLGALVYSNSAVNPILYAFLSENFRKSFFKAFTCMNKQDINAQLQLEPSVFTKQGSKKRG GSKRLLTSNPQIPPLLPLNAGNNNSSTTTSSTTTAEKTGTTGTQKSCNSNGKVTAPPENLIICLSE QQEAFCTTARRGSGAVQQTDL

DmGPCR9 ( SEQ ID NO：21 ) DNA ATGTTCAACTACGAGGAGGGGGATGCCGACCAGGCGGCCATGGCTGCAGC GGCTGCCTATAGGGCACTGCTCGACTACTATGCCAATGCGCCAAGTGCGG CGGGTCACATAGTGTCGCTCAACGTGGCACCCTACAATGGAACTGGAAAC GGAGGCACTGTCTCCTTGGCGGGCAATGCGACAAGCAGCTATGGCGATGA TGATAGGGATGGCTATATGGACACCGAGCCCAGTGACCTGGTCACCGAAC TGGCCTTCTCCCTGGGCACCAGTTCAAGTCCAAGTCCCAGTTCCACACCC GCTTCCAGCTCCAGTACTTCCACTGGCATGCCCGTCTGGCTGATACCCAG CTATAGCATGATTCTGCTGTTCGCCGTGCTGGGCAACCTGCTGGTCATCT CGACGCTGGTGCAGAATCGCCGGATGCGTACCATAACCAACGTGTTCCTG CTCAACCTGGCCATATCGGACATGCTGCTGGGCGTGCTCTGCATGCCCGT CACCCTGGTGGGCACCCTGCTGCGAAACTTCATCTTTGGCGAGTTCCTCT GCAAGCTCTTTCAGTTCTCGCAAGCCGCCTCCGTGGCCGTTTCGTCCTGG ACCTTGGTGGCCATATCCTGTGAGCGCTACTACGCGATATGCCATCCACT GCGCTCGCGATCCTGGCAGACAATCAGTCACGCCTACAAGATCATCGGCT TCATCTGGCTGGGCGGCATCCTCTGCATGACGCCCATAGCGGTCTTTAGT CAATTGATACCCACCAGTCGACCGGGCTACTGCAAGTGCCGTGAGTTTTG GCCCGACCAGGGATACGAGCTCTTCTACAACATCCTGCTGGACTTCCTGC TGCTCGTCCTGCCGCTTCTCGTCCTCTGCGTGGCCTACATCCTCATCACG CGTACCCTGTACGTAGGCATGGCCAAGGACAGCGGACGCATCCTGCAGCA ATCGCTGCCTGTTTCCGCTACAACGGCCGGCGGAAGCGCACCGAATCCGG GCACCAGCAGCAGTAGTAACTGCATCCTGGTCCTGACCGCCACCGCAGTC TATAATGAAAATAGTAACAATAATAATGGAAATTCAGAGGGATCCGCAGG CGGAGGATCAACCAATATGGCAACGACCACCTTGACAACGAGACCAACGG CTCCAACTGTGATCACCACCACCACGACGACCACGGTGACGCTGGCCAAG ACCTCCTCGCCCAGCATTCGCGTCCACGATGCGGCACTTCGCAGGTCCAA CGAGGCCAAGACCCTGGAGAGCAAGAAGCGTGTGGTCAAGATGCTGTTCG TCCTGGTGCTGGAGTTTTTCATCTGCTGGACTCCGCTGTACGTGATCAAC ACGATGGTCATGCTGATCGGACCGGTGGTGTACGAGTATGTCGACTACAC GGCCATCAGTTTCCTCCAGCTGCTGGCCTACTCATCCAGCTGCTGCAATC CGATCACCTACTGCTTCATGAACGCCAGCTTCCGGCGCGCCTTTGTCGAC

ACCTTCAAGGGTCTGCCCTGGCGTCGTGGAGCAGGTGCCAGCGGAGGCGT CGGTGGTGCTGCTGGTGGAGGACTCTCCGCCAGCCAGGCGGGCGCAGGCC CGGGCGCCTATGCGAGTGCCAACACCAACATTAGTCTCAATCCCGGCCTA GCCATGGGTATGGGCACCTGGCGGAGTCGCTCACGCCACGAGTTTCTCAA TGCGGTGGTGACCACCAATAGTGCCGCCGCCGCCGTCAACAGTCCTCAGC TCTA ( SEQ ID NO：22 ) SEQ ID NO：21DNA ： MFNYEEGDADQAAMAAAAAYRALLDYYANAPSAAGHIVSLNVAPYNGTGNGGTVSLAGNATSSYGD DDRDGYMDTEPSDLVTELAFSLGTSSSPSPSSTPASSSSTSTGMPVWLIPSYSMILLFAVLGNLLV ISTLVQNRRMRTITNVFLLNLAISDMLLGVLCMPVTLVGTLLRNFIFGEFLCKLFQFSQAASVAVS SWTLVAISCERYYAICHPLRSRSWQTISHAYKIIGFIWLGGILCMTPIAVFSQLIPTSRPGYCKCR EFWPDQGYELFYNILLDFLLLVLPLLVLCVAYILITRTLYVGMAKDSGRILQQSLPVSATTAGGSA PNPGTSSSSNCILVLTATAVYNENSNNNNGNSEGSAGGGSTNMATTTLTTRPTAPTVITTTTTTTV TLAKTSSPSIRVHDAALRRSNEAKTLESKKRVVKMLFVLVLEFFICWTPLYVINTMVMLIGPVVYE YVDYTAISFLQLLAYSSSCCNPITYCFMNASFRRAFVDTFKGLPWRRGAGASGGVGGAAGGGLSAS QAGAGPGAYASANTNISLNPGLAMGMGTWRSRSRHEFLNAVVTTNSAAAAVNSPQL

DmGPCR10 ( SEQ ID NO：23 ) DNA ATGTACGCCTCCTTGATGGACGTTGGCCAGACGTTGGCAGCCAGGCTGGCGGATAGCGAC GGCAACGGGGCCAATGACAGCGGACTCCTGGCAACCGGACAAGGTCTGGAGCAGGAGCAG GAGGGTCTGGCACTGGATATGGGCCACAATGCCAGCGCCGACGGCGGAATAGTACCGTAT GTGCCCGTGCTGGACCGCCCGGAGACGTACATTGTCACCGTGCTGTACACGCTCATCTTC ATTGTGGGAGTTTTGGGCAACGGCACGCTGGTCATCATCTTCTTTCGCCACCGCTCCATG CGCAACATACCCAACACATACATTCTTTCACTGGCCCTGGCTGATCTGTTGGTTATATTG GTGTGTGTACCTGTGGCCACGATTGTCTACACGCAGGAAAGCTGGCCCTTTGAGCGGAAC ATGTGCCGCATCAGCGAGTTCTTTAAGGACATATCCATCGGGGTGTCCGTGTTTACACTG ACCGCCCTTTCCGGCGAGCGGTACTGCGCCATTGTAAATCCCCTACGCAAGCTTCAGACC AAGCCGCTCACTGTCTTTACTGCGGTGATGATCTGGATCCTGGCCATCCTACTGGGCATG CCTTCGGTTCTTTTCTCCGACATCAAGTCCTACCCTGTGTTCACAGCCACCGGTAACATG ACCATTGAAGTGTGCTCCCCATTTCGCGACCCGGAGTATGCAAAGTTCATGGTGGCGGGC AAGGCACTGGTGTACTACCTGTTGCCGCTGTCCATCATTGGGGCGCTATACATCATGATG GCCAAGCGGCTCCATATGAGCGCCCGCAACATGCCCGGCGAACAGCAGAGCATGCAGAGC CGCACCCAGGCTAGGGCCCGACTCCATGTGGCGCGCATGGTGGTAGCATTCGTGGTGGTG TTCTTCATCTGCTTCTTCCCGTACCACGTGTTTGAGCTGTGGTACCACTTCTACCCAACG GCTGAGGAGGACTTCGATGAGTTCTGGAACGTGCTGCGCATCCTTCCTAAACTCGTGCGT CAACCCCGTGGCCTCTACTGCGTGTCCGGGGTGTTTCGGCAGCACTTTAATCGCTACCTC TGCTGCATCTGCGTCAAGCGGCAGCCGCACCTGCGGCAGCACTCAACGGCCACTGGAATG ATGGACAATACCAGTGTGATGTCCATGCGCCGCTCCACGTACGTGGGTGGAACCGCTGGC AATCTGCGGGCCTCGCTGCACCGGAACAGCAATCACGGAGTTGGTGGAGCTGGAGGTGGA GTAGGAGGAGGAGTAGGGTCAGGTCGTGTGGGCAGCTTTCATCGGCAGGACTCGATGCCC CTGCAGCACGGAAATGCCCACGGAGGTGGTGCGGGCGGGGGATCCTCCGGACTTGGAGCC GGCGGGCGGACGGCGGCAGTGAGCGAAAAGAGCTTTATAAATCGTTACGAAAGTGGCGTA ATGCGCTACTAA ( SEQ ID NO：24 ) SEQ ID NO：23DNA ： MYASLMDVGQTLAARLADSDGNGANDSGLLATGQGLEQEQEGLALDMGHNASADGGIVPYVPVLDR PETYIVTVLYTLIFIVGVLGNGTLVIIFFRHRSMRNIPNTYILSLALADLLVILVCVPVATIVYTQ ESWPFERNMCRISEFFKDISIGVSVFTLTALSGERYCAIVNPLRKLQTKPLTVFTAVMIWILAILL GMPSVLFSDIKSYPVFTATGNMTIEVCSPFRDPEYAKFMVAGKALVYYLLPLSIIGALYIMMAKRL HMSARNMPGEQQSMQSRTQARARLHVARMVVAFVVVFFICFFPYHVFELWYHFYPTAEEDFDEFWN VLRILPKLVRQPRGLYCVSGVFRQHFNRYLCCICVKRQPHLRQHSTATGMMDNTSVMSMRRSTYVG GTAGNLRASLHRNSNHGVGGAGGGVGGGVGSGRVGSFHRQDSMPLQHGNAHGGGAGGGSSGLGAGG RTAAVSEKSFINRYESGVMRY

According to budapest treaty, clone of the present invention is deposited in the international depositary institution of agricultural research typical case's culture (NRRL), 1815 N.University Street, and Peoria, Illinois 61604, U.S.A.Following table 5 provides accession number and preservation date.

The clone	The NRRL accession number	Preservation date
			??DmGPCR1(SEQ?ID?NO：1)	??NRRL?B-30347	On October 19th, 2000
??DmGPCR2a(SEQ?ID?NO：3)	??NRRL?B-30348	On October 19th, 2000
			??DmGPCR4(SEQ?ID?NO：7)	??NRRL?B-30349	On October 19th, 2000
??DmGPCR5a(SEQ?ID?NO：9)	??NRRL?B-30350	On October 19th, 2000
			??DmGPCR6aL(SEQ?ID?NO：13)	??NRRL?B-30351	On October 19th, 2000
??DmGPCR6bL(SEQ?ID?NO：15)	??NRRL?B-30352	On October 19th, 2000
			??DmGPCR7(SEQ?ID?NO：17)	??NRRL?B-30353	On October 19th, 2000
??DmGPCR8(SEQ?ID?NO：19)	??NRRL?B-30354	On October 19th, 2000
			??DmGPCR9(SEQ?ID?NO：21)	??NRRL?B-30355	On October 19th, 2000

The present invention also is used to illustrate embodiments of the invention and is illustrated by following.These embodiment be not for, or be interpreted into and limit the scope of the invention.Should know that the present invention can implement by the mode except this paper specifically describes.According to the instruction of this paper, can carry out many modifications of the present invention and change, therefore within the scope of the invention.

Each patent, application and the printed publication of mentioning in this patent file in this complete introducing for your guidance.

Embodiment

The evaluation of embodiment 1:DmGPCR

Convert Celera genotype fruit bat database the protein and the mRNA database of prediction to range gene search software instrument, the mRNA (" PnuFlyPep " database) that will produce with prediction.The method of gene search software tool analysis genome database is well known by persons skilled in the art.

With the nucleotide sequence of several nematodes (C.elegans) FaRP GPCR as search sequence to above-mentioned mRNA database.Use various tool, comprise that FASTA and breach BLAST retrieve the zone that has similarity in this database (Altschul etc., Nuc.Acids Res., 1997,25,3389, in this complete introducing for your guidance).

Simply say, represent the BLAST algorithm of basic local arrangements contrast gopher to be suitable for measuring sequence similarity (Altschul etc., molecular biology magazine 215:403-410 (1990), for your guidance) in this complete introducing.By biotechnology infonation center (http://www.ncbi.nlm.nih.gov/)The public can obtain to carry out the software that BLAST analyzes.This algorithm comprises and is tested and appraised at first that length is the word of W in the search sequence, identifying the pairing (HSP) of high scoring, when with database sequence in during the word arrangement contrast of same length, mate or satisfy certain on the occasion of threshold scoring T.T is called adjacent words scoring threshold value (Altschul etc. are on seeing).Find these initial adjacent words as the starting point that causes the search of seeking the longer HSP that contains them.With the word that finds along the two-way extension of each sequence, up to the arrangement contrast scoring that can increase accumulative total.When: the arrangement contrast scoring of (1) accumulative total reaches value whereabouts quantity X from its maximum; (2) because one or more negative scoring residues are arranged the accumulative total of contrast, this accumulative total mark reach 0 or below; Or (3) when reaching one of two sequences terminal, and the word that finds extends and is stopped.BLAST algorithm parameter W, T and X have determined to arrange the sensitivity and the speed of contrast.BLASTN program (being used for nucleotide sequence) is 11 as the sentence long (W) of default value, use the BLOSUM62 rating matrix (to see Henikoff etc., Proc.Natl.Acad.Sci.USA 89:10915-10919 (1989)) arranging (B) is 50, desired value (E) is 10, M=5, N=-4, and relatively more double-stranded.

BLAST algorithm (Karlin etc., Proc.Natl.Acad.Sci.USA 1993,90:5873-5787, in this complete introducing for your guidance) and breach BLAST algorithm also carry out the statistical analysis of similarity between two sequences.A kind of similarity measurement method that the BLAST algorithm provides is minimum possibility sum (P (N)), and it provides the indication of possibility, by it coupling between two nucleotide or the amino acid sequence can take place by chance.For example, if in test nucleic acid and comparison DmGPCR nucleic acid, minimum possibility sum is less than about 1, preferably more less than about 0.1, is more preferably less than about 0.01 and most preferably less than about 0.001, nucleic acid is considered as similar to DmGPCR gene or cDNA.

From the prediction mRNA database retrieval that is used to prepare the PnuFlyPep database to corresponding to the mRNA of predicted protein matter.These are accredited as following nucleotide sequences: SEQ ID NOs:1, and 3,5,7,9,11,13,15,17,19,21 and 23, all have significant overlapping homology on the statistics with this search sequence.Nucleotide sequence SEQ IDNOs:3,5,9,11,13 and 15 (corresponding to DmGPCRs 2a, 2b, 5a, 5b, 6a, and 6b) identify the order-checking of sequence (not shown) available from the PCR clone with to another.Every splice variant of all representing the DmGPCR gene of these sequences.

The clone of embodiment 2:DmGPCR

The cDNA preparation

From fruit bat (Drosophila melanogaster) the poly A that grows up ⁺RNA (Clontech Laboratories, PaloAlto, CA) or the total RNA of adult fruit bat (hereinafter) preparation cDNA.In order to obtain total RNA, (Biological Supply Company, Burlington NC), are containing 10ml H to the parental generation original seed of crymoanesthesia fruit bat ₂Add the only adult fruit bat of 5-6 in O, 10mlFormula4-24 Instant Drosophila nutrient culture media and 6-10 grain active dry yeast (Biological SupplyCompany) culture tube.The terminal isocyanurate foam bolt of placing of each test tube, room temperature is cultivated fruit bat 4-6 week.In the maturity stage, freezing this test tube is poured the fruit bat of anesthesia in the 50ml polypropylene test tube that is contained in the liquid nitrogen into.Freezing fruit bat is stored at-70 ℃, up to grinding them with mortar, and smashs to pieces with pestle in the presence of liquid nitrogen.Powdered tissue and some liquid nitrogen are strained in the 50ml polypropylene test tube on the dry ice.After the liquid nitrogen vaporization, powdered tissue is stored at-70 ℃.

In order to prepare RNA, the 300mg powdered tissue is placed polypropylene test tube on the dry ice, add the 0.1M NaOAc of 5ml6M guanidine hydrochloride, pH5.2 solution.Handle all solution with DEPC, or, dry all glasswares, or use and do not have used plastics testing chamber equipment, to reduce the Rnase pollution problems with the dH2O preparation that DEPC-handles.The test tube rotation mixes, and places on ice then.With passing through 20,21, No. 22 syringe needle homogenization powdered tissue continuously.Centrifuge tube (100xg10min) is layered on the 2.5-3ml supernatant then and is contained in 14 * 95mmUltra Clear centrifuge tube (Beckman Instruments, Inc., Palo Alto, CA) top of middle 8ml5.7M cesium chloride 0.1M NaOAc solution.Under 18 ℃, sample the L8-70 ultracentrifuge (BeckmanInstruments, Inc.) in 25000rpm centrifugal 18 hours.The decantation supernatant is inverted test tube and is dried.With the flaky precipitate of RNA be suspended in 200 microlitres and do not have the dH2O of Rnase (Qiagen Inc., Valencia, CA) in, do not have twice of the dH2O drip washing (totally 400 microlitres) of Rnase then with 100 microlitres.Add 44 microlitre 3M NaOAc, the cold 100% precipitation with alcohol RNA of pH5.2 and 1ml.-70 ℃ of storages that spend the night, 14000rpm centrifuge tube 1 hour (Eppendorf microcentrifugal tube 5402), with 75% ethanol (dH2O that handles with DEPC prepares) drip washing, precipitation is dissolved in the dH2O of no Rnase.At 10mM Tris-HCl, detect 260 or the absorbance of 280nm among the pH7.5, be used to estimate concentration and the purity of RNA.

(method that MD) provides prepares the first chain cDNA for GIBCO BRL, Rockville according to Superscript II enzyme.In the microcentrifugal tube that contains the dH2O that has or not Rnase and 250ng (2.5 microlitre) random primer, add 500ng (2 microlitre) poly A ⁺The total RNA of RNA or 3 micrograms (4 microlitre).Test tube (12 microlitre) was cultivated 10 minutes at 70 ℃, and cooled on ice adds 4 microlitre 5x, the first chain damping fluid, 2 microlitre 0.1M DTT and 1 microlitre 10mMdNTP potpourri then.25 ℃ 10 minutes, 42 ℃ cultivated in 2 minutes after, add 1 microlitre (200 unit) Superscript II, continue to cultivate 50 minutes at 42 ℃.Cultivate for 70 ℃ and stopped enzyme reaction in 15 minutes.In order to remove the RNA with the cDNA complementation, (Boehringer Mannheim, Indianapolis IN), cultivated 20 minutes for 37 ℃ then to add 2 microlitres (2 unit) Rnase H.CDNA is stored at-20 ℃.

The PCR reaction

With the 50/100 microlitre PCR reaction or the heat start PCR reaction of standard, use Ampliwax pearl (PerkinElmer Cetus, Norwalk, CT) amplification fruit bat g protein coupled receptor (DmGPCR).With the distilled water solution primer (Genosys Biotechnologies, Inc., The Woodlands, TX): 510 μ M concentration '-and 3 '-primer, the middle primer of 1 μ M.Each PCR reactant contains the rTth XL of 2-4 unit archaeal dna polymerase, 1.2-1.5mMMg (OAc) ₂, 200 each dNTP of μ M and 200 or each primer of 400nM.For heat start PCR, each 2-4 microlitre primer adds 32 or 36 microlitre " downstream " potpourri (dH2O, 3.3x XL-damping fluid, dNTP and Mg (OAc) ₂) (cumulative volume 40 microlitres).Add Ampliwax pearl (Perkin Elmer Cetus), cultivated test tube 5 minutes for 75 ℃, the room temperature cooling adds 60 microlitres " upstream " potpourri (dH2O, 3.3x XL-damping fluid, rTth and template) then.In perkin Elmer Series 9600 thermal cyclers, carry out pcr amplification.The exemplary program of thermal cycler comprises: 94 ℃ 1 minute, be then 30 take turns amplification (94 ℃ 0.5 minute, 60 ℃ 0.5 minute, 72 ℃ 2 minutes), be then 60 ℃ 6 minutes.In order on the PCR product, to set up 3 ' A-jag (" tailing "), when pcr amplification finishes, add 1 microlitre Taq polymerase (Invitrogen, Carlsbad, CA).Cultivated test tube 10 minutes for 72 ℃.Analyze reaction mixture in 1% Ago-Gel of TAE damping fluid (5) preparation.Usually with QIAquick post (QIAGEN) purified pcr product that spins.

Connect and conversion

All PCR products are connected into PCR3.1 carrier (Invitrogen),, the product that connects is transformed into One Shot according to the explanation of manufacturer ^TMTOP10F ' competent cell (Invitrogen).Cultivate the transformant that will screen embolus at the LB meat soup that contains 50 microgram ampicillin/ml.With boiling-the little prepared product method of cracking plasmid (5) or identify colony with embolus by direct " colony PCR " method (6) from the bacterium amplified plasmid dna that transforms.

Dna sequencing

With Qiagen anion exchange plasmid kit (QIAGEN-tip20), from the 5ml LB nutrient culture media of 37 ℃ of overnight incubation, indicate DNA isolation, the DNA that preparation will be checked order according to manufacturer.Usually adopt four kinds of primers (T7, M13 are reverse, " justice is arranged " and " antisense ") (table 6) each DNA that checks order.Adopt dyestuff-terminator order-checking chemical reagent, i.e. BigDye ^TMTerminator reagent (Applied Biosystems, Foster City, CA) or DYEnamic ^TMET terminator kit (Amersham Pharmacia Biotech, Inc., Piscataway, NJ).According to the recommendation of manufacturer, carry out sequencing reaction.Spinning with Centri-Sep, (Princeton Separations, Adelphia NJ) remove primer and uncorporated nucleotide to post.On Applied Biosystems 377 robotization dna sequencing instrument, analyze the sequencing reaction thing.With Sequencher (Gene Codes, Ann Arbor, MI), sequential analysis program (WisconsinPaekage Version 10.1, science of heredity computing machine group (GCG), Madison, WI) GCG assembling is joined and the analyzing DNA sequence, and by (the program suit of Vector NTI 5.5 MD) makes the DNA functionating for Informax, Bethesda.

Table 6 dna sequencing primer

DmGP CR	5 ' primer	3 ' primer	Middle primer
					Justice is arranged	Antisense
			1	?VGS28-gtagccgccATGGCC ?AACTTAAGCTGGCTGA ?GCAC(SEQ?ID?NO：184)			VGS29-gtaTCAGTTGATT CGCCTCCCCAGCTCT (SEQ?ID?NO：185)	VGS49-TGCAGCATCTAC ATATCCACGCTGA(SEQ ID?NO：186)	VGS50-GATTGGCG ACACGGCACCCGT GCCA(SEQ?ID NO：187)
2	?VGS30-gtagccgccATGTCA ?CTACCCAGCTGGCTAAC ?AGA ?(SEQ?ID?NO：188) ?DEL1937-gccgccATGAAT ?CAGACGGAGCCCGCCC ?AGC ?(SEQ?ID?NO：189)	VGS31gtaTTACCGCGGC ATCAGCTTGGTGACC (SEQ?ID?NO：190)			VGS59-GTACGGCGTGCT AATCGTCTTCGGC(SEQ ID?NO：191)	VGS60-ATTGCGAG CAGTGCGCATGAT GGGC(SEQ?ID NO：192)

????3	??DEL1840-gccgccATGTCG ??GAGATTGTCGACACCG ??AGC(SEQ?ID?NO：193)	??DEL1860-TTCCAGTGGC ??AGGACAGATCGGGAT ??(SEQ?ID?NO：194)	VGS65-ATGTGGCCAGAT GGACGATATCCCA(SEQ ID?NO：195)	??VGS66-CAATCATG ??GGAATGCCCGTAG ??TCAG(SEQ?ID ??NO：196)
					????4	??DEL1933-gccgccATGGAG ??AACACCACAATGCTGG ??CTA(SEQ?ID?NO：197)	??DEL1934-TTAGAGTCCA ??GTGGTGGAGGTCCTG ??(SEQ?ID?NO：198)	VGS47-GCCATCATCCGG CCACTGCAGCCGC(SEQ ID?NO：199)	??VGS48-AATGGGAT ??TGTACATGGAGTT ??GCTC(SEQ?ID?NO： ??200)
????5	??DEL1844-gccgccATGGAG ??AATCGCAGTGACTTCG ??AGGC(SEQ?ID?NO：201)	??DEL1845-tctagaTCAGGAG ??AGCAGTTGGGTGGTGTT ??GGC(SEQ?ID?NO：202)	DEL1891-ATCTCCATCG ACAGATACGT(SEQ?ID NO：203)	??DEL1892-GCCGCGA ??TGGCCAGGTTGCA ??(SEQ?ID?NO：204)
					????6	??DEL1842-gccgccATGTAC ??TACATAGCTCACCAGC ??AGCCG(SEQ?ID?NO：205) ??DEL1990-gccgccATGGAG ??CACCACAATAGCCATCT ??GTT(SEQ?ID?NO：206)	??DEL1862-CGATCGGCGC ??ACCGGAGAATCAGTT ??(SEQ?ID?NO：207) ??DEL1989-TCAAAACTCG ??GTGCTTCTTATGTTTG ??(SEQ?ID?NO：208)	VGS51-GTCACCAATTAC TTTATAGCCAGCT(SEQ ID?NO：209)	??VGS52-GGGCAGCC ??AACAGCAGGTGAA ??CACA(SEQ?ID ??NO：210) ??DEL1991-GTGAGAT ??GACTACGAAGTAC ??CATC(SEQ?ID ??NO：211)
????7	??VGS69-gtagccgccATGGCA ??ATGGACTTAATCGAGC ??A(SEQ?ID?NO：212)	??VGS70-TTAAAGTGGTTG ??CCACAAGGACT(SEQ?ID ??NO：213)	VGS74-GGGCACACGTG CTCCTGGTAACG(SEQ ID?NO：214)	??VGS73-ATAGAGCT ??GCAGTGGCAGCCA ??GC(SEQ?ID?NO：215)
					????8	??VGS38-gtagccgccATGTTT ??ACGTGGCTGATGATGG ??ATGT(SEQ?ID?NO：216)	??VGS39-gtaATTACAAATC ??TGTCTGCTGCACTGCG ??(SEQ?ID?NO：217)	VGS55-GTGCAAAGCCT ACATGGTGAGCACA (SEQ?ID?NO：218)	??VGS56-TGAGTATTT ??CCAGTCGGGAGAG ??GTC(SEQ?ID ??NO：219)
????9	??VGS40-gtagccgccATGTTC ??AACTACGAGGAGGGGG ??ATGC(SEQ?ID?NO：220)	??VGS41-gtaTTAGAGCTGA ??GGACTGTTGACGGCG ??(SEQ?ID?NO：221)	VGS53-GTGCTCTGCATG CCCGTCACCCTGG(SEQ ID?NO：222)	??VGS54-GACGAACA ??GCATCTTGACCAC ??ACGC(SEQ?ID ??NO：223)
					????11	??DEL1905-gccgccATGGCT ??GGCCATCAGTCGCTGG ??CAC(SEQ?ID?NO：224)	??DEL1906-TTAGAGCATT ??TCAATATTGGACGTT ??(SEQ?ID?NO：225)	VGS57-CCCGTGACTAGC ATGTCCCTGCGAA(SEQ ID?NO：226)	??VGS58-ACCGGAAT ??CGCAGTCGTCACA ??ATCG(SEQ?ID ??NO：227)

The result of DmGPCR clone of the present invention and order-checking is as follows:

DmGPCR1

With design corresponding to the amplification of the PCR primer success of the cDNA of PnuFlyPep34651 from fruit bat polyA ⁺The PCR product of the cDNA prepared product of mRNA preparation.The product that obtains is cloned and checked order.The sequence that test obtains is identical with forecasting sequence.Obtain complete clone, be called DmGPCR1.

DmGPCR2

With being designed to corresponding to the initial trial of the PCR primer amplification PCR product of the cDNA of PnuFlyPep67585 and unsuccessful.With this forecasting sequence and existing nematode acceptor and other neuropeptide receptor arrangement contrast, show 5 ' terminal unusual length of forecasting sequence, prompting may be wrong in the predictive genes of this side.As guide, designed and tested 5 ' PCR primer of various alternatives with genome sequence.One of these combination of primers are used from the product that has obtained having just size of the cDNA success of total RNA preparation.Clone derived from the PCR reaction is checked order, show that amplified production comprises 5 ' and 3 ' end of prediction, identical with forecasting sequence, except forecasting sequence lacks 6 amino acid whose extensions.Relatively the clone has also disclosed and has had two kinds of montage isoforms, and a kind of similar to forecasting sequence (being called DmGPCR2a), another lacks to be positioned at and passes through 23 amino acid extensions (being called DmGPCR 2b) that TM VIII enters the born of the same parents C end of molecule.

DmGPCR3

Reported gene in the document corresponding to DmGPCR predicted protein matter.This gene (GenBank accession number M77168) is known as NKD, " a kind of tachykinin receptor of developmental regulation ".Monnier D, etc., J.Biol.Chem.1992,267 (2), 1298-302.Relatively M77168 shows that with the PnuFlyPep68505 sequence sequence of prediction is significantly different with cDNA.CDNA has 5 long ' end, lacks 51 amino acid whose extrons of a coding, and obviously shorter at 3 ' end.To the sequences Design PCR primer of publishing, with each cDNA acquisition PCR product of total RNA system.This product is identical with the structure of the NKD sequence of report.

DmGPCR4

Use PCR primer corresponding to the cDNA design amplification DmGPCR4 of PnuFlyPep67393.CDNA library with total fruit bat mRNA preparation obtains and has cloned the PCR product.Compare the sequence of these clones and PnuFlyPep prediction, it is identical to disclose sequence, and an extron of predicting except HMMGene is not present in any clone's the PCR product.DmGPCR4 is recently by Lenz etc., Biochem.Biophys.Res.Comm., and 2000,273,571-577 etc. clone, and are categorized into the allatostatin acceptor of second supposition.

DmGPCR5

DmGPCR5 (FlyPepCG7887) mistake contain frameshift mutation.The PnuFlyPep version, PnuFlyPep67522 (be called in the literature recently " the fruit bat acceptor of tachykinin related peptides " (M77168) (Li XJ, etc., EMBO Journal, 1991,10 (11), 3221-3229)), corrected this mistake, but wrong prediction some internal sequences and C-end.At first sight, the cDNA corresponding to the prediction of PnuFlyPep protein is identical with reported sequence.Use the PCR primer, from fruit bat poly A ⁺The amplification of the cDNA potpourri success of mRNA preparation the PCR product of correct size.PCR product order-checking to the clone discloses, though whole splice mode is identical, has two montage mistakes in the PnuFlyPep sequence.These mistakes cause frameshift mutation, are the frameshift mutation of a compensation subsequently, and causing has 13 amino acid whose differences from 46 in amino acid between sequence test determination and report.This cloned genes is called DmGPCR5a.

In addition, found the montage isoform of DmGPCR5.The terminal extracellular domain of this variant coding N-has 3 extra amino acid.This variant is called as DmGPCR5b.

DmGPCR6

GPCR corresponding to PnuFlyPep15731 be described as in the literature " neuropeptide tyrosine " acceptor (M81490.Li XJ, etc., J.Biol.Chem., 1992,267 (1), 9-12).The sequence and the M81490 of PnuFlyPep prediction are inequality at the molecule two ends.PnuFlyPep15731 compares with M81490 at the N end has 15 extra amino acid.3 ' end of PnuFlyPep 15731 is also different with M81490, is brachymemma, does not contain conservative TM VI and TM VII residue.

With the initial PCR primer of the sequences Design of M81490.With these primers with from the template of total mRNA, obtained the PCR product.The detection of clone's PCR product discloses, and it uses the tupe identical with M81490.This clone is called as " DmGPCR6a ".

In the process of clone DmGPCR6a, found extra montage isoform.Produce another 3 ' end of molecule with acceptor splicing site alternately, use identically, produced this isoform but have different sequences of reading frames with the 6a major part.In addition, open reading frame extend through 3 ' PCR primer of this clone.Detect 3 ' terminal genome sequence and disclosed many possible candidate's extrons.Tested PCR primer, up to finding to increase the PCR product corresponding to these possible extrons.This product is called 6b.As if the detection of this genome sequence has also been predicted the initiation codon ATG of PnuFlyPep 15731 and has contained 15 extra amino acid whose M81490 initiation codons in same reading frame that PnuFlyPep 15731 initiation codons are real initiation codons.Designed new 5 ' PCR primer, wherein added PnuFlyPep 15731 initiation codons, be used in combination, increased and cloned DmGPCR6aL and DmGPCR6bL (length) with two 3 ' PCR primers.

DmGPCR7

The initial trial of amplification DmGPCR7 gene outcome is also unsuccessful.This forecasting sequence (PnuFlyPep 67863) is arranged contrast with other GPCR, and prompting has mistake in the prediction of molecule 3 ' end.3 ' terminal other GPCR of ratio great majority of forecasting sequence are much longer.The possible mistake of detection prompting of genome sequence occurs in the montage prediction of removing in-frame stop codon, and this terminator codon should obtain the molecule of just size.Designed 3 ' PCR primer in this introne.In addition, designed 5 ' new PCR primer, to utilize just ATG in the frame of the upstream from start codon of predicting.Pcr amplification obtains having the product of desired size from the cDNA of total mRNA.

The N-end of the PnuFlyPep of DmGPCR7 and WO01/70980 version all lacks two amino acid.As previously mentioned, PnuFlyPep and FlyPep CG10626 version are all incorrect at the C-end.Predict that incorrect DmGPCR7 gene outcome version is the fruit bat leucokinin acceptor inferred (Hewes ﹠amp for example; Taghert, Genome Res., 2001,11,1126-1142; Holmes etc., Insect Mol.Biol., 2000,9,457-465).Yet, before the present invention, do not have experimental evidence to confirm this supposition.

DmGPCR8

With the DmGPCR8 that successfully increased of the PCR primer to the sequences Design of PnuFlyPep prediction.Use the template as the PCR reaction derived from the cDNA of poly A+RNA.Structurally all the sequence with the PnuFlyPep prediction is identical for the clone of whole 6 order-checkings.Noticed polymorphism at 68 (dna sequence dnas), wherein half to be cloned in this position be " C ", half is " A ".This change causes amino acid change, is respectively Asp or Glu.The Celera sequence is designated as " A ", and therefore optional " A " clone (Glu) is used for further research.Do not obtain correctly towards " A " clone, therefore carried out utilizing the subclone step of Pme I to remove insertion from original pCR3.1 clone, the pCR3.1 carrier that digests with Pme I reverse its towards.

The PnuFlyPep version is correct.Yet WO01/70980 lacks about 17-terminal amino acids and about 15 internal amino acids.This receptor is categorized into somatostatin sample acceptor (the Hewes ﹠amp for example of supposition; Taghert, Genome Res., 2001,11,1126-1142).Before this invention, there is not experimental evidence to confirm this supposition.

DmGPCR9

With the PCR primer that the PnuFlyPep forecasting sequence is designed and from poly A ⁺The cDNA template clone DmGPCR9 of RNA preparation.In PnuFlyPep, correctly predicted genome structure.

DmGPCR10

The initial trial that produces the PCR product with the primer of DmGPCR10 (PnuFlyPep70325) design is unsuccessful.The detection of the cDNA of prediction shows that the sequence of prediction is uncommon, because it does not contain " WXP " motif among the TM VI of high conservative, does not also contain " NPXXF " motif of TMVII, though there are several other conserved residues.The genome sequence that detects last extron downstream 80kb does not disclose any other possible extron.Do not attempt obtaining the complete clone of DmGPCR10.

DmGPCR11 (allatostatin sample peptide acceptor)

PCR primer with the sequences Design " allatostatin sample peptide acceptor " of publishing.Birgul etc., EMBOJournal, 1999,18 (21), 5892-5900.Use cDNA to obtain the PCR product, clone and order-checking derived from total mRNA prepared product.The final cDNA of coded protein is described identical with publication.

Embodiment 3:RNA engram analysis

Can carry out rna blot analysis, detect the expression of mRNA.As mentioned above with have justice towards oligonucleotides and antisense towards oligonucleotides as primer, the part that increased is selected from SEQ ID NO:1,3,5,7,9,11,13,15,17, the GPCR cDNA sequence of 19,21 and 23 nucleotide sequence.

A plurality of people from Clontech (people II#7767-1) organize RNA trace and probe hybridization.Prehybridization is at 42 ℃, 5xSSC, and 1x Denhardt ' s reagent, 0.1%SDS, 50% formamide carried out in the 250mg/ml salmon sperm dna 4 hours.Hybridization in same potpourri 42 ℃ spend the night and carry out, add about 1.5 * 10 ⁶The probe of cpm/ml mark.

With α-32P-dCTP label probe, go up purifying by Rediprime dna marker system (Amersham Pharmacia), be added in the hybridization solution at Nick post (Amersham Pharmacia).Under 42 ℃, in 0.2xSSC and 0.1%SDS, wash filter membrane several times.Under-80 ℃, (N.Y. USA) goes up exposure for Eastman Kodak Company, Rochester at Kodak XAR film to make filter membrane with the enhancing mask.

Embodiment 4: recombinant expressed DmGPCR in eukaryotic

In mammalian cell, express DmGPCR

In order to prepare DmGPCR albumen, the technique for gene engineering with suitable expression vector and standard in proper host cell is expressed the DmGPCR polynucleotide encoding.For example, the sequence subclone of embodiment 1 described DmGPCR coding is gone into commodity expression vector pzeoSV2 (Invitrogen, San Diego, CA), be transfected into Chinese hamster ovary cell (CHO) with transfection reagent FuGENE6 (Boehringer-Mannheim), the transfection scheme is provided in the product inset.Other eukaryotic cell lines comprises that for example human embryo kidney (HEK) (HEK293) and COS cell also are suitable.By (CA) there are cultivation down in Stratagene, LaJolla, select the cell of stably express DmGPCR at 100 mcg/ml zeocin.Optional, the available standards chromatographic technique is from cell purification DmGPCR.In order to promote purifying, generation is corresponding to the antiserum of one or more synthetic property peptide sequences of the part of DmGPCR amino acid sequence, with this antiserum affinity purification DmGPCR.DmGPCR also can express in frame with tailer sequence (for example polyhistidyl, hemagglutinin, FLAG), to promote purifying.In addition, will know many purposes of DmGPCR polypeptide, for example following test does not need from host cell purifying DmGPCR.

In 293 cells, express DmGPCR

For the expression of DmGPCR in 293 cells, prepared the plasmid that carries relevant DmGPCR coded sequence with carrier pSecTag2A (Invitrogen).Carrier pSecTag2A contains the mouse IgK chain targeting sequencing that is useful on secretion, available anti--the c-myc epi-position of myc antibody test recombinant protein, be used for the terminal polyhistidyl of C-of nickel chelating chromatography and be used to select the zeocin resistant gene of stable transfectant.Measure the forward primer of this GPCR cDNA by conventional method, preferably contain 5 of nucleotide ' extension, with introduce the HindIII cloning site and with the nucleotide of GPCR sequences match.Also detected reverse primer, preferably contained 5 ' nucleotide and extend, the XhoI restriction site that is used to clone with introducing and corresponding to the nucleotide of the reverse complemental thing of DmGPCR sequence with conventional method.The PCR condition is that annealing temperature is 55 ℃.Gel-purified PCR product is cloned into the HindIII-XhoI site of carrier.

With Qiagen chromatographic column purify DNA, (Boehringer Mannheim, Indianapolis IN) are transfected into 293 cells with the DOTAP transfection media.After 24 hours, carry out western blot analysis in transfection, the expression of test transient transfection cell with anti-His and anti--DmGPCR peptide antibody.Select permanent transfectional cell and propagation with Zeocin.By carrying out western blot analysis, from cell and nutrient culture media, all detect the generation of recombinant protein with anti--His, anti--Myc or anti--GPCR peptide antibody.

The expression of DmGPCR in the COS cell

In order in the COS7 cell, to express DmGPCR, can be selected from SEQ ID NO:1 with containing, 3,5,7,9,11,13,15,17,19,21, or the polynucleotide molecule of 23 nucleotide sequence is cloned among the carrier p3-CI.This carrier is the plasmid that pUC18 derives, and wherein contains HCMV (human cytomegalovirus) promoter-be positioned at introne and a multiple clone site of bGH (bovine growth hormone) polyadenylic acid sequence upstream.In addition, this plasmid contains dhfr (dihyrofolate reductase) gene, and it can select stable transformant in the presence of medicine amethopterin (MTX).

Determining forward primer with conventional method, preferably contain 5 ' extend, with the XbaI restriction site that introducing is used to clone, is corresponding to being selected from SEQ ID NOs:1 then, 3,5,7,9,11,13,15,17,19,21, or the nucleotide of 23 nucleotide sequence.Also having determined reverse primer with conventional method, preferably contained 5 of nucleotide ' extension, introduced the SalI cloning site, is corresponding to SEQ ID NOs:1 subsequently, 3,5,7,9,11,13,15,17,19,21, and the reverse complemental thing of o or 23 nucleotide sequence.

PCR is included in 95 ℃ of initial denaturing steps of 5 minutes, and 30 take turns, and 95 ℃ of sex change in 30 seconds were annealed 30 seconds and extended 30 seconds at 72 ℃, be to extend 5 minutes at 72 ℃ subsequently for 58 ℃.This PCR product connects into XbaI and the SalI site of carrier p3-CI by gel-purified.This construction is transformed into Bacillus coli cells, is used for amplification and DNA purifying.With Qiagen chromatographic column purify DNA, be transfected into the COS7 cell according to manufacturers instruction with the Lipofectamine reagent of BRL.Transfection 48 and after 72 hours, the recombinant protein expression in test media and the cell.

Can then by for example chromatographic purifying protein, come the DmGPCR of purifying COS cell culture expression by cell growth medium being concentrated to about 10mg protein/ml.The DmGPCR of purifying is concentrated to 0.5mg/ml in the Amicon thickener of YM-10 film is housed, be stored at-80 ℃.

The expression of DmGPCR in the insect cell

In order to express DmGPCR in rhabdovirus system, available pcr amplification is selected from SEQ ID NO:1, and 3,5,7,9,11,13,15,17,19,21, or the polynucleotide molecule of 23 nucleotide sequence.Determining forward primer with conventional method, preferably contain and add 5 of NdeI cloning site ' extension, is corresponding to being selected from SEQ ID NO:1 then, 3,5,7,9,11,13,15,17,19,21, or the nucleotide of 23 nucleotide sequence.Also having determined reverse primer by conventional method, preferably had and introduce 5 of KpnI cloning site ' extend, is corresponding to being selected from SEQ ID NO:1 subsequently, 3,5,7,9,11,13,15,17,19,21, or the nucleotide of the reverse complemental thing of 23 nucleotide sequence.

Gel-purified PCR product with NdeI and KpnI digestion, is cloned into carrier pAcHRL-A (Pharmingen, San Diego, corresponding site CA).The pAcHTL expression vector contains the strong poly-polyhedrin promoter of Autographa californica nuclear polyhedrosis virus (AcMNPV) and at the 6Xhis of multiple clone site upstream tail.Also exist for the protein kinase site of phosphorylation with in order before multiple clone site, to downcut the fibrin ferment site of recombinant protein.Certainly, also available many other baculovirals replace pAcHTL-A, for example pAc373, pVL941 and pAcIM1.Also can use other to express the suitable carriers of GPCR polypeptide, condition is as needs, and this vector construction should comprise the positioning signal of transcribing, translating and transport, for example AUG and signal peptide in the frame.These carriers such as Luckow etc., Virology 170:31-39 etc. are described.

With standard baculovirus expression method, as (A MANUAL OF METHODS FORBACULOVIRUS VECTORS AND INSECT CELL CULTURE PROCEDURES, Texas AgriculturalExperimental Station Bulletin No.1555 (1987)) described cultivation and isolated virals such as Summers.

In one embodiment, (CA) method of using manufacturer to provide will contain the pAcHLT-A introducing baculoviral of DmGPCR gene for Pharmingen, San Diego with " BaculoGold " transfection reagent box.After transfection 24 hours, with the protein production of the cell analysis individual cells separator of the radiolabeled infection of 35S-methionine.Infect and collected the cell that infects in back 48 hours, with the albumen of SDS-PAGE show tags.The virus of separable demonstration high expression level, and be used for extensive expression.

In order to express the DmGPCR polypeptide in the Sf9 cell, available above-mentioned primer and the method that is used for baculovirus expression carried out PCR, and amplification has the SEQ of being selected from ID NO:1, and 3,5,7,9,11,13,15,17,19,21, or the polynucleotide molecule of 23 nucleotide sequence.DmGPCR cDNA is cloned among the carrier pAcHLT-A (Pharmingen), in the Sf9 expressed in insect cells.After removing inner NdeI site (with the identical primer of above-mentioned baculovirus expression) this embolus is cloned into NdeI and KpnI site.With Qiagen chromatographic column purify DNA, in the Sf9 cell, express.Tested the recombinant protein that reacts with the GPCR specific antibody during the preliminary Western blotting of purifying plaque is tested with expection size.After in the HIG5 cell, being further purified and expressing optimization, these results have been confirmed.

Embodiment 5: interact and catch/two-hybrid system

In order to test the DmGPCR action protein, available this interaction is caught/double cross library screening method.This test is at first at Fields, etc., Nature, describes in 1989,340,245, in this complete introducing for your guidance.At CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley ﹠amp; Sons, NY, 1999 and Ausubel etc., SHORT PROTOCOLS IN MOLECULAR BIOLOGY, the 4th edition, Greene andWiley-interseience, NY has published a kind of method in 1992, in this complete introducing for your guidance.Kit can be available from Clontech, Palo Alto, CA (Matchmaker two-hybrid system 3).

In suitable plasmid (being pGBT7) with standard subclone technique construction coding all or part DmGPCR and yeast transcription factor GAL4 DNA fusions in conjunction with the nucleotide sequence of territory (DNA-BD).Similarly, (method that forms the cDNA library is seen Sambrook etc. to have made up fusion library, the active territory of GAL4 (AD) in second plasmid (being pGADT7) from the possible protein-bonded cDNA of GPCR, MOLECULARCLONING:A LABORATORY MANUAL, second edition, Cold Spring Harbor Press, Cold SpringHarbor, NY, 1989, in this complete introducing for your guidance).By sequence verification the DNA-BD/GPCR fusion constructs, tested activation of autonomous reporter and cytotoxicity, the both can stop the double cross analysis.Similarly control with AD/ library fusion constructs, guarantee the expression in the host cell and lack transcriptional activity.With GPCR and library fusion plasmid according to standard method (Ausubel etc., SHORT PROTOCOLS IN MOLECULARBIOLOGY, the 4th edition, Greene and Wiley-interscience, NY, 1992, in this complete introducing for your guidance) transformed yeast cell (about 105 transformants/mg DNA).DNA-BD/GPCR causes transcribing of specific yeast plasmid reporter (being lacZ, HIS3, ADE2, LEU2) with interior combination of the body of AD/ library albumen.Yeast cells is seeded on the auxotrophy nutrient culture media expression of screening reporter.After detection is grown in the nutrient culture media that is supplemented with Xgal (5-bromo-4-chloro-3-indyl-β-D-galactoside), (the filter membrane test of betagalactosidase activity is as Breeden etc. for twice of the betagalactosidase activity of colony, Cold Spring Harb.Symp.Quant.Biol., 1985,50, described in 643, for your guidance) in this complete introducing.Save positive AD library plasmid from transformant, introduce original yeast strains again and contain the bacterial strain of irrelevant DNA-BD fusion, confirm specific DmGPCR/ library albumen effect with other.The DNA that inserts is checked order, the existing of the open reading frame that merges with checking and GAL4 AD, and detect the protein-bonded identity of DmGPCR-.

Embodiment 6: carry out mobility shifting DNA-in conjunction with test with gel electrophoresis

Gel electrophoresis mobility shifting test detection specificity protein-DNA effect rapidly.Method extensively can get in handbook, Sambrook etc. for example, MOLECULAR CLONING:A LABORATORY MANUAL, second edition, Cold Spring Harbor Press, Cold Spring Harbor, NY, 1989 and Ausubel etc., SHORTPROTOCOLS IN MOLECULAR BIOLOGY, the 4th edition, Greene and Wiley-interscience, NY, described in 1992 handbooks such as grade, for your guidance in this complete introducing.From synthetic property oligonucleotides, restriction enzyme enzyme fragment or PCR fragment obtain dna probe (＜300bp), terminal using ³²The P mark.Under equilibrium temperature (22-37 ℃), at the 10-15 microlitre damping fluid that contains radiolabeled dna probe, non-specific carrier DNA (about 1 microgram), BSA (300 μ g/ml) and 10% (v/v glycerine) (is TAE or TBE, pH8.0-8.5) in, cultivated the DmGPCR (about 15 micrograms) of an equal portions purifying or thick DmGPCR extract (about 15ng) at least 30 minutes.Then reaction mixture is added on the polyacrylamide gel,, well separates with protein-DNA compound up to free dna probe in the 30-35mA operation.Desiccant gel then detects band corresponding to dissociative DNA and protein-DNA compound with radioactive automatic developing.

Embodiment 7:DmGPCR antibody

Polyclone or monoclonal antibody with standard technique generation DmGPCR produce its useful antibodies fragment or its variant, comprise " humanization " variant.These methods can be in (1989) such as for example Sambrook, see above and Harlow etc. (volumes) ANTIBODIES:A LABORATORY MANUAL, Cold Spring HarborLaboratory, Cold Spring Harbor, NY, discovery in 1988.In one embodiment, the reorganization DmGPCR polypeptide cell or the cell membrane of these polypeptide (or contain) is as the antigen that produces antibody.In another embodiment, with having one or more peptides corresponding to the amino acid sequence of the immunogenicity of DmGPCR part (for example 6,7,8,9,10,11,12,13,14,15,16,17,18,19,20 or more a plurality of amino acid) as antigen.Corresponding to the peptide of the outer part of DmGPCR born of the same parents, the outer part of especially hydrophilic born of the same parents belongs to the present invention.Antigen can mix with adjuvant, or is connected with haptens, to increase antibody producing.

Polyclone or monoclonal antibody

As exemplary method, with reorganization DmGPCR or its synthetic fragment immune mouse, the generation monoclonal antibody (or bigger mammal, for example rabbit obtains polyclonal antibody).In order to improve antigenicity, peptide and keyhole  hemocyanin (Pierce) are recommended coupling according to manufacturer.Injection first, antigen Freund's complete adjuvant emulsification, hypodermic injection.Two to three weeks of interval are with the DmGPCR antigen of other equal portions of incomplete Freund emulsification, hypodermic injection.The last time before the booster shots, from the mouse acquisition blood serum sample of inoculation, with the existence of Western blotting assessment with the immunoreactive antibody of DmGPCR.Can be used to the serum of animal from inoculation as polyclonal antiserum or be used to separate the polyclonal antibody of discerning DmGPCR.In addition, can kill mouse, take off its spleen and be used to produce monoclonal antibody.

In order to produce monoclonal antibody, spleen is placed 10ml serum-free RPMI 1640, by being supplemented with 2mM L-glutaminate, 1mM Sodium Pyruvate, 100 units/ml penicillin and 100 μ g/ml streptomysin (RPMI) (Gibco, Canada) grind spleen among the serum-free RPMI 1640, form single cell suspension.The filtration cell suspension, centrifuge washing is suspended among the serum-free RPMI again.Prepare thymocyte in a similar manner, as feeder layer from three non-immune Balb/c mouse.Logarithmic phase NS-1 myeloma cell before merging, placed in 3 days be supplemented with 10% hyclone (FBS (and Hyclone Laboratories, Inc., Logan, Utah) among) the RPMI, centrifugal and abundant washing.

In order to produce the hybridoma fusion, the spleen cell and the NS-1 mixing with cells of immune mouse is centrifugal, and inhale and remove supernatant.Pat test tube cell precipitation is come off, will precipitate with 2ml37 ℃ of PEG1500 (75mM HEPES, 50% solution among the pH8.0) and (Boehringer-Mannheim) stir, add serum-free RPMI then.Then, centrifuge cell, be suspended in again contain 15%FBS, 100 μ M hypoxanthine, 0.4 μ M aminopterin-induced syndrome, 16 μ M thymines (HAT) (Gibco), 25 units/ml IL-6 (Boehrnger-Mannheim) and 1.5 * 10 ⁶Among the RPMI of thymocyte/ml, be layered in 1O the flat 96 hole tissue culturing plates of Corning (Corning, Corning New York).

After fusion the 2nd, 4 and 6 day, from merge dull and stereotyped hole, inhale and remove 100 microlitre nutrient culture media, replace with fresh culture.The 8th day, screen fusions with ELISA, whether test exists the existence of the mouse IgG that can combine with DmGPCR.By diluting the selected fusion of further clone hole, up to the monoclonal culture that obtains to produce anti--DmGPCR antibody.

The humanization of anti--DmGPCR monoclonal antibody

The expression pattern of DmGPCR as described herein and GpCR intervene certified track record as treatment and have pointed out the treatment indication of DmGPCR inhibitor (antagonist).The DmGPCR neutralizing antibody is the therapeutic agent of a class as the DmGPCR antagonist.Be humanization monoclonal antibody method of the present invention below.

Humanized principle has described in the literature, and is subjected to the module arrangement promotion of antibody protein.In order to reduce the possibility of conjugated complement as far as possible, can use the humanized antibody of IgG4 isotype.

For example, can contain the variable region of interested non-human antibody's albumen and the chimeric antibody of human antibody molecules constant region by generation, realize certain humanization level (see for example Morrison etc., Adv.Immunol., 1989,44,65-92).From the genomic DNA of B cell hybridoma or from separate the cDNA clone DmGPCR neutrality that produces from the mRNA of hybridoma interested anti--variable domain of DmGPCR antibody.V district genetic fragment is connected with the extron of coding people antibody constant region, and the construction that obtains is expressed in proper host cell (for example myeloma or Chinese hamster ovary celI).

In order to realize even higher levels of humanization, only the part of the variable region gene fragment of non-human monoclonal antibodies gene code antigen-binding complementary determining region (" CDR ") is cloned into human antibody sequence and (sees for example Jones etc., Nature, 1986,321,522-525; Riechmann etc., Nature, 1988,332,323-327; Verhoeyen etc., Science, 1988,239,1534-36; With Tempest etc., Bio/Technology, 1991,9,266-71).As needs, the beta sheet framework that people's antibody is surrounded the CDR3 district is modified into more (sees Kettleborough etc., Protein Engin., 1991,4,773-783 near the three-dimensional structure of the initial monoclonal antibody antigen binding domain of mirror image mapping; With Foote etc., J.Mol.Biol., 1992,224,487-499).

In another approach,, for example come the interested non-human monoclonal antibodies of humanization surface, keep all inside of this non-human antibody and contact residues simultaneously by direct mutagenesis by changing the selected surface residue of non-human antibody.See Padlan, Molecular Immunol., 1991,28 (4/5), 489-98.

Carry out said method with anti-GPCR monoclonal antibody of DmGPCR neutrality and their hybridoma of generation, thereby produced humanization DmGPCR neutrality antibody, be used for the treatment of or alleviate the expression of DmGPCR or the disease that ligand-mediated DmGPCR signal transmission plays illeffects as therapeutic agent.

Embodiment 8: identify the test of the correctives of DmGPCR activity

Be several nonrestrictive tests that are used to identify the correctives (activator and antagonist) of DmGPCR activity below.In the correctives that can identify by these tests, the native ligand compound of acceptor, the synthetic analogues and the derivant of native ligand are arranged; Antibody; Antibody fragment; And/or derived from natural antibody or from the antibody sample compound of antibody sample combinatorial libraries; And/or the synthetic property compound that identifies of the high flux screening by the library etc.The correctives that combines with DmGPCR is used for appraisement organization's sample DmGPCR of (for example being used for diagnostic purpose, pathology purpose etc.).Activator and antagonist correctives are respectively applied for to be in harmonious proportion reduces the DmGPCR activity.An available correctives of inferring.And/or available known activator combination candidate antagonist (vice versa) is carried out this test.

The cAMP test

In class test, in contacting the DmGPCR cells transfected of candidate modulator compound, measure the level of cyclisation adenine one phosphoric acid (cAMP).The method that cAMP analyzes is described to some extent in the literature and (is for example seen Sutherland etc., Circulation, 1968,37,279; Frandsen etc., Life Sciences, 1976,18,529-541; Dooley etc., J.Pharm.and Exper.Ther., 1997,283 (2), 735-41; With George etc., J.Biomolecular Screening, 1997,2 (4), 235-40).Hereinafter list the exemplary method of these tests, used NEN ^TMThe adenylate cyclase enzyme activation FlashPlate  test of Life Science Products.

Simply say, DmGPCR coded sequence (for example genomic DNA of DNA or intronless) subclone is gone into commercially available expression vector, pzeoSV2 (Invitrogen) for example, use known method, for example the transfection method that provides of Boehringer-Mannheim is gone into Chinese hamster ovary cell (CHO) with this expression vector transient transfection when the FuGENE6 transfection reagent is provided.The Chinese hamster ovary celI of transfection is inoculated 96 hole microtiter plates into FlashPlate  assay kit, and this plate is combined with the cAMP antiserum thereon with solid scintillator parcel.In contrast, some hole inoculations have wild type (untransfected) Chinese hamster ovary celI, and the cAMP standard solution of different amounts is accepted in other hole in the flat board, is used to set up typical curve.

In the cell in each hole, add one or more test compounds (being candidate modulator), water and/or do not have compound nutrient culture media/thinning agent in contrast.After the processing, make cAMP at room temperature, in cell, assembled just in time 15 minutes.Add and to contain [ ¹²⁵I]-the cAMP lysis buffer termination test of mark, use Packard Topcount ^TM96 hole microtiter plate scintillation counters are counted flat board.The unlabelled cAMP of cell lysis (or standard items) and fixed amount [ ¹²⁵I]-antibody that combines on cAMP competition and the flat board.Make up typical curve, obtain unknown cAMP value by interpolation.Response engaged test compound, the change of cAMP level has indicated DmGPCR to regulate activity in the cell born of the same parents.As with the G Protein G _sThe correctives of hypotype coupled receptor agonists is with cAMP production, and the 3-10 that causes the cAMP level to measure doubly increases.With the G Protein G _I/oThe cAMP that the activator of hypotype coupled receptor will suppress forskolin and stimulate produces, and the 50-100% that causes the cAMP level to measure reduces.To reverse these effects of the acceptor that activates to the composition activation or by known activator as the correctives of inverse agonist.

The aequorin test

In another experiment, use the construction transient cotransfection cell (for example Chinese hamster ovary celI) of DmGPCR expression constructs and encoded luminescent albumen aequorin.In the presence of the co-factor coelenterazine, aequorin will be launched measurable light, (kytoplasm) free calcium amount is proportional in itself and the born of the same parents (sees for example Cobbold, Deng, " Aequorinmeasurements of cytoplasmic free calcium, " is in CELLULAR CALCIUM:A PRACTICALAPPROACH, McCormack J.G. and Cobbold P.H., compile Oxford:IRL Press, 1991; Stable etc., Anal.Biochem., 1997,252,115-26; And Haugland, HANDBOOK OF FLUORESCENT PROBESAND RESEARCH CHEMICALS, sixth version, Eugene, or, Molecular Probes, 1996).

In an exemplary test, the DmGPCR subclone is gone into commercially available expression vector pzeoSV2 (Invitrogen), with construction (the Molecular Probes of transfection reagent FuGENE 6 (Boehringer-Mannheim) with encoded luminescent albumen aequorin, Eugene, OR), the transfection scheme transient cotransfection that provides with the product inset.

At the MEM (Gibco/BRL that is supplemented with 10% hyclone, 2mM glutamine, 10U/ml penicillin and 10 μ g/ml penicillin and 10 μ g/ streptomysins, Gaithersburg, MD) 37 ℃ of cultured cells are 24 hours in, this moment nutrient culture media is replaced with and contains 5 μ M coelenterazine (Molecular Probes, Eugene, serum-free MEM OR).Continue then to cultivate 2 hours at 37 ℃.Subsequently, make cell detachment, wash, and be suspended in again among the serum-free MEM with 200,000 cells/ml with VERSEN (Gibco/BRL).

In serum-free MEM, prepare the dilution of candidate DmGPCR correctives compound, and be dispersed in the Zhu Kongzhong of opaque 96 hole breadboards with 50 μ l/ holes.Then flat board is installed to MLX microtiter plate luminescence assays instrument (Dynex Technologies, Inc., Chantilly, VA) on.This instrumentation program is set and 50 μ l cell suspensions can be dispersed in each hole, and every hole is surveyed once, reads luminosity immediately 15 seconds.The dosage that makes up candidate modulator with each light signal peak area under curve relies on curve.Use the formula analysis data of a site part with SlideWrite, obtain EC ₅₀Value.Think that the change of the luminosity that compound causes indicated the correctives activity.As with the G Protein G _qThe correctives of the activator of the acceptor of hypotype coupling makes luminosity improve to reach 100 times.To reverse this effect of composition acceptor active or that activated by known activator as the correctives of inverse agonist.

The luciferase reporter gene test

The luminescent protein luciferase provides the useful tool of the correctives of another kind of analysis DmGPCR activity.With DmGPCR expression constructs (for example DmGPCR among the pzeoSV2) be included in the transcription factor binding site point, the report construction transient cotransfection cell of the gene of the luciferase protein in cAMP response element (CRE), AP-1 or NF-κ B downstream (for example Chinese hamster ovary celI or COS7 cell) for example.Can with the G Protein G _sThe activator of hypotype coupled receptor combination can cause cAMP to increase, thereby the activating ELK 1 transcription factor causes the expression of luciferase gene.Can with the G Protein G _qThe activator of hypotype coupled receptor combination can cause producing DG, thus activator protein matter kinase c, and it can activate the AP-1 or the NF-κ B factor, thereby causes the expression of luciferase gene.The luciferase expression level has reflected that signal transmits the state of activation of incident.Usually see George etc., J.Biomolecular Screening, 1997,2 (4), 235-240; With Stratowa etc., Curr.Opin.Biotechnol., 1995,6,574-581.Available for example Promega (Madison, the luciferase test reagent quantitative measurement uciferase activity of WI) buying.

In an exemplary test, in transfection before 1 day, the density of Chinese hamster ovary celI with 100,000 cells/well is layered in the 24 hole double dish, in the MEM (Gibco/BRL) that contains 10% hyclone, 2mM glutamine, 10U/ml penicillin and 10 μ g/ml streptomysins, cultivates for 37 ℃.With DmGPCR expression constructs and the report construct while transient cotransfection cell that contains luciferase gene.Report plasmid CRE-luciferase, AP-1-luciferase and NF-κ B-luciferase can available from Stratagene (LaJolla, CA).(Boehringer-Mannheim) carries out transfection according to manufacturers instruction with the FuGENE6 transfection reagent.Available report construct cells transfected in contrast.After the transfection 24 hours, with pre-temperature once, in cell, add serum-free MEM (contrast) then or contain the serum-free MEM of one or more candidate modulator, 37 ℃ of cultured cells 5 hours to 37 ℃ PBS washed cell.Then, with ice-cold PBS washed cell once, 100 μ l lysis buffer cracking of the luciferase assay kit that provides from Promega are provided in every hole.Room temperature was cultivated after 15 minutes, the potpourri in opaque and white 96 orifice plates with 15 microlitre lysates and 50 microlitre substrate solutions (Promega), immediately at Wallace1450 type MicroBeta flicker and luminescent counter (Wallace Instruments, Gaithersburg reads luminosity on MD) immediately.

Luminosity difference when existing and not having the candidate modulator compound shows that the correctives activity is arranged.Composition activates or is compared with the reporter cells transfected with single by the activator activated receptors, and 3-20 times stimulating light emission is arranged.Correctives as inverse agonist reverses this effect.

Use FLIPR to measure the cellular calcium level

The variation of cellular calcium level is the index that the another kind of G-G-protein linked receptor activity has been familiar with, and this test can be used for screening the correctives of DmGPCR activity.For example, will be with the Chinese hamster ovary celI of DmGPCR expression vector stable transfection with 4 * 10 ⁴The density of cells/well is seeded in 96 orifice plates of Packard black wall, and the special design of this flat board is used for distinguishing the dull and stereotyped fluorescence signal that send in each hole of going up.Cellular incubation is containing 36mg/L pyruvic acid and 1g/L glucose and four kinds of calcon-carboxylic acid dyestuff (Fluo-3 ^TMAM, Fluo-4 ^TMAM, Calcium Green ^TM-1AM or Oregon Green ^TMOne of 488 BAPTA-1 AM), respectively be among the improvement Dulbecco ' sPBS (D-PBS) of 4 μ M concentration 37 ℃ 60 minutes.Cultivated 10 minutes for dull and stereotyped twice, 37 ℃ with improvement D-PBS washing, remove remaining dyestuff from cell membrane.In addition, before activating the calcium reaction, carry out a series of washing with improvement D-PBS immediately.

Add one or more candidate receptor agonist compounds, calcium ion carrier A 23187 (10 μ M, positive control) or ATP (4 μ M, positive control) and cause the calcium reaction.With the FLIPR of the MolecularDevice with argon laser (exciting) at 488nm measure fluorescence (for example see Kuntzweiler etc., Drug Dev.Res., 1998,44 (1), 14-20).The F-aperture of detector camera is decided to be 2.5, and exposure length is 0.4 millisecond.Before adding candidate's activator, ATP or A23187, measure the basic fluorescence 20 seconds of cell, the basic fluorescence level of deduction from reaction signal.Measure about 200 seconds of calcium signal, carried out single reading in per two seconds.Calcium ion carrier A 23187 and ATP bring up to more than the baseline values 200% with the calcium signal.Usually, the GPCR of activation brings up to about 10-15% more than the background signal with the calcium signal.

The mitosis test

In mitosis test, measured candidate modulator induce or suppress the fissional ability of DmGPCR mediation (for example see Lajiness etc., J.Pharm.and Exper.Ther., 1993,267 (3), 1573-1581).For example, the Chinese hamster ovary celI of stably express DmGPCR is seeded in 96 orifice plates with the density of 5000 cells/well, cultivates 48 hours in the MEM that is supplemented with 10% hyclone for 37 ℃, uses twice in serum-free MEM drip washing cell this moment.After the drip washing, add the fresh MEM of 80 microlitres, or contain the MEM of known mitogen, and 20 microlitres contain one or more candidate modulator of the variable concentrations that dilutes or the MEM of test compounds in serum free medium.In contrast, serum free medium is only accepted in some holes on each flat board, and the nutrient culture media of 10% hyclone is accepted to contain in some holes.The cell of untransfected or independent suppressed by vector cells transfected also can be used as contrast.

Cultivate after 16-18 hour, in the hole, add 1 μ Ci [ ³H]-thymine (2Ci/mmol), cultivated cell again 2 hours for 37 ℃.Trypsin digestion and cell is collected on the filter paper pads of cell harvesting instrument (Tomtec); In the Betaplate counter, filter paper is counted then.With mix in the serum-free instrument connection [ ³H]-result of thymine and serum stimulation cell (positive control) is relatively.With the test compounds of multiple concentration, use nonlinear least square fitting formula: A=B * [C/ (D+C)]+G to set up and the analysis dose-effect curve, wherein A is a serum stimulation percentage; B is that maximum efficiency subtracts baseline; C is EC ₅₀D is a compound concentrations; G is a maximum efficiency.B parameter, C and G optimize mensuration by Simplex.

Estimate activator with receptors bind can improve intracellular [ ³H]-thymine mixes, shows seroreaction is improved 80%.The antagonist that can combine with this receptor will suppress the being seen stimulation of known activator, reach 100%.

[ ³⁵S] GTP γ S is in conjunction with test

Because g protein coupled receptor transmits signal by G albumen in the born of the same parents, and the activity of G albumen comprises that GTP combination and hydrolysis produce the GDP of combination in the born of the same parents, measure the GTP analog that exists and do not have non-hydrolysable under the candidate modulator situation [ ³⁵S] GTP γ S, provide another kind of correctives activity test (see for example Kowal etc., Neuropharmacology, 1998,37,179-187).

In an exemplary test, in 10cm tissue culture ware, grown into the Asia by the cell of DmGPCR expression vector stable transfection and converge, with the ice-cold no Ca of 5ml ²⁺/ Mg ²⁺The phosphate-buffered salt rinsing once scrapes in the identical damping fluid of 5ml.Centrifugal (500xg, 5 minutes) sedimentation cell is resuspended in the TEE damping fluid (25mM Tris, pH 7.5,5mM EDTA, 5mM EGTA), and liquid nitrogen is freezing.After the thawing, with Dounce homogenizer homogenate cell (every plate cell 1ml TEE), 1, centrifugal 5 minutes of 000xg removes stoning and not broken cell.

20, centrifugal homogenate supernatant is 20 minutes under the 000xg, the diffusion barrier component, and with TEE washing film precipitation once, (20mM HEPES, pH 7.5,150mM NaCl, 10mM MgCl to be resuspended in binding buffer liquid ₂, 1mMEDTA) in.Can be in liquid nitrogen the film of freezing resuspension, and be stored under-70 ℃, up to use.

Melt the equal portions prepare and be stored at-70 ℃ cell membrane as mentioned above, homogenate, and go into to contain 20mM HEPES, 10mM MgCl with 10-50 μ g/ml concentration dilution ₂, 1mM EDTA, 120mM NaCl is in the damping fluid of 10 μ MGDP and 0.2mM ascorbic acid.Homogenate was cultivated 30 minutes for GTP30 ℃ with the final volume of 90 microlitres and the candidate modulator compound or the 100 μ M of variable concentrations, placed on ice then.For each sample, add 10 microlitre guanines 5 '-O-(3[ ³⁵S] sulphur) triphosphoric acid (NEN, 1200Ci.mmol; [ ³⁵S]-GTP γ S) the realization ultimate density is 100-200pM.Sample was cultivated 30 minutes at 30 ℃ again, and 4 ℃ add 1ml10mMHEPES, pH7.4,10mM MgCl ₂, reaction stops by filtering.

Filtered sample on Whatman GF/B filter membrane, with the ice-cold 10mM HEPES of 20ml, pH 7.4,10mM MgCl ₂Washing.With the liquid scintillation photometric analysis filter membrane is counted.In the presence of 100 μ M GTP, measure [ ³⁵S]-non-specific binding of GTP γ S, from sum, deduct.Select to compare with the untransfected control cells, regulate [ ³⁵S]-compound of GTP γ S binding capacity in cell.Activator to the activation of acceptor cause [ ³⁵S]-GTP γ S is in conjunction with increasing by 500.Antagonist this reaction of blockading.

The map kinase activity test

The activity of map kinase provides the test method of the correctives of another kind of evaluation DmGPCR activity in the cell of assessment expression GPCR.See for example Lajiness etc., J.Pharm.and Exper.Ther., 1993,267 (3), 1573-1581 and Boulton etc., Cell, 1991,65,663-675.

In one embodiment, tested before 48 hours, will inoculate in 6 orifice plates with 70,000 cells/well with the Chinese hamster ovary celI of DmGPCR stable transfection.During these 48 hours, 37 ℃ of cultured cells in the MEM nutrient culture media that is supplemented with 10% hyclone, 2mM glutamine, 10U/ml penicillin and 10 μ g/ streptomysins.Before adding stimulant, first serum starvation cell 1-2 hour.

For test, handle cell separately or with the nutrient culture media that contains candidate's activator or 200nM phorbol ester-acetate myristin (being PMA, positive control) with nutrient culture media, cell is cultivated different time down at 37 ℃.In order to stop this reaction, flat board is layered on ice, inhales and removes nutrient culture media, with the ice-cold PBS drip washing of the 1ml cell that contains 1mM EDTA.Then, (12.5mM MOPS, pH 7.3,12.5mM phosphoglycerol, 7.5mM MgCl to add 200 microlitre cell lysis buffer solution in cell ₂, 0.5mM EGTA, 0.5mM sodium vanadate, 1mM benzenecarboximidamide, 1mM dithiothreitol (DTT), 10 μ g/ml leupeptins, 10 μ g/ml aprotinins, 2 μ g/ml pepstatin A and 1 μ M arachidonic acid).Cell is scraped from flat board, carry out homogenate 10 times by 23 3/4G syringe needles, and 20,000xg preparation in centrifugal 15 minutes cell liquid component.

With cytosol equal portions (the 5-10 microlitre that contains the 1-5 micrograms of protein) and 1mM MAPK peptide substrate (APRTPGGRR (SEQ ID NO:168), Upstate Biotechnology, Inc., N.Y.) and 50 μ M[γ- ³²P] (NEN 3000Ci/mmol) mixes ATP, and being diluted to is about 2000cpm/pmol than vigor finally, and cumulative volume is 25 microlitres.Sample was cultivated 5 minutes at 30 ℃, at 2cm ²Whatman P81 cellulose phosphate paper on point sample 20 microlitres, cessation reaction.Wash the filter paper square 4 times with 1%H3PO4, square is carried out the liquid scintillation spectrophotometric analysis, quantitatively the label of combination.The cytosol extract of equivalent is not cultivated with the MAPK peptide substrate, the binding label of these samples is deducted from the coupling sample of handling with peptide substrate.The cytosol extract in each hole can be used as burble point.(BioRad Laboratories) measures protein concentration in conjunction with test with protein dye.Estimate that activator causes the increase of MAPK enzymatic activity to reach 5 times to the activation of this receptor.Antagonist this increase of can blockading.

[ ³H] arachidonic acid release

The activation of observing GPCR has strengthened the arachidonic acid in the cell and has discharged, and the useful test of another kind of GPCR active regulator is provided.See for example Kanterman etc., Molecular Pharmacology, 1991,39,364-369.For example, will be by the Chinese hamster ovary celI of DmGPCR expression vector stable transfection with 15,000 cells/well is layered on 24 orifice plates, and in the MEM nutrient culture media that is supplemented with 10% hyclone, 2mM glutamine, 10U/ml penicillin and 10 μ g/ streptomysins 37 ℃ of cultured cells 48 hours, use then.With 0.5 μ Cl/ml [ ³H] (Amersham Corp. 210Ci/mmol) is supplemented with 10mM HEPES at 1ml to arachidonic acid, and 37 ℃ of each porocytes of mark are 2 hours in the MEM nutrient culture media of the bovine serum albumin of pH7.5 and 0.5% FAF.Use twice of the identical damping fluid washed cell of 1ml then.

The candidate modulator compound is added in the 1ml same buffer separately or with 10 μ M ATP, cultivated cell 30 minutes for 37 ℃.Only use damping fluid and simulation cells transfected in contrast.Carry out liquid scintillation spectrophotometric counting with 0.5ml sample to every hole.The activator of activated receptor cause that ATP stimulates [ ³H]-arachidonic acid release enhancing.This enhancing is sealed by antagonist.

Extracellular acidification speed

In another experiment, change the effect of the candidate modulator of having analyzed the DmGPCR activity by the outer pH of the born of the same parents that induce in the monitoring test compounds.See for example Dunlop etc., J.Pharmacological and ToxicologicalMethods, 1998,40 (1), 47-55.In one embodiment, will be by the Chinese hamster ovary celI of DmGPCR expression vector transfection with 4 * 10 ⁵Cell/micella is inoculated in the 12mm capsule shells (Molecular Devices Corp.) in the MEM nutrient culture media that contains 10% hyclone, 2mM glutamine, 10U/ml penicillin and 10 μ g/ streptomysins.In this nutrient culture media, at 5%CO ₂Cultivated 24 hours for following 37 ℃.

Measure extracellular acidification speed with the micro-physiological function detector of Cytosensor (Molecular Devices Corp.).Capsule shells is contained in the detecting chamber of little physiological detection instrument, this chamber is with the mobile damping fluid (MEM of no carbonic acid hydrogen salt is supplemented with 4mM L-glutaminate, 10U/ml penicillin, 10 μ g/ streptomysins and 26mM NaCl) of flow velocity 100 μ l/min perfusion.Candidate's activator or other reagent are by mobile damping fluid dilution, and perfusion is by second fluid passage.During per 60 seconds pumping circulation, in 38 seconds of pump operation, all the other 22 seconds are out of service.The pH of running buffer in the record detecting chamber in the 43-58 circulation of second.Restart the circulation of beginning next round then at 60 seconds pumps.Calculate the acidification rate of the damping fluid that in writing time, flows with the Cytosoft program.Deduct the change that baseline value (for adding the average of correctives material standed for 4 velocity determination values before) calculates acidification rate from adding the flank speed measured value that obtains behind certain correctives material standed for.Selected instrument measures 61mV/pH unit.Speed when causing the extracellular acidification speed ratio not have activator as the correctives of this receptor activator improves.This reaction is used as the correctives of receptor antagonist and blockades.

Embodiment 9: DmGPCR and peptide part are complementary

Cell culture and transfection

Containing 5%CO ₂The moistening atmosphere of air in, be supplemented with in the complete DMEM nutrient culture media of 10% hot deactivation FBS, the huge mycin of 10 μ g/ml, 0.1M nonessential amino acid 37 ℃ and cultivate wild type Chinese hamster ovary cells (CHO-K1) (from American type culture collection, Rockville, MD) or the CHO-10001A cell.Use LipofectAMINE PLUS ^TMAccording to manufacturers instruction, with the pCR3.1 carrier transfectional cell that contains orphan GPCR DNA.Simply say, with Chinese hamster ovary celI be layered on 10cm sterilization the tissue culture ware (Corning GlassWorks, Corning, NY) in, on transfection same day, cell reaches 50-60% and converges.In plastic test tube, formerly be diluted in adding PLUS (20 microlitres/plate) in the cDNA plasmid (5 micrograms/plate) among the 0.75ml OptiMEM, mix also and at room temperature cultivated 15 minutes.In addition, LipofectAMINE (30 microlitres/plate) is mixed with 0.75mlOptiMEM, be added in the DNA/PLUS potpourri that is pre-mixed, at room temperature cultivated 15 minutes.Nutrient culture media with on serum-free transfection media (common DMEM, 5ml/ plate) the replacement cell adds DNA-PLUS-LipofectAMINE compound (1.5ml/ plate), and gentleness is mixed in the nutrient culture media, then at 37 ℃/5%CO ₂Under cultivated 3 hours.In nutrient culture media, add the complete DMEM nutrient culture media that contains 20%FBA (6.5ml/ plate) then, continue at 37 ℃/5%CO ₂Under cultivated 24-48 hour.In Chinese hamster ovary celI, carry out moment GFP with the plasmid (GFP, 4 micrograms/plate) of green fluorescent protein and express, be evaluated at the used the same terms of GPCR under the productive rate of transfection.

Film preparation

With ice-cold Dulbecoo phosphate-buffered salt (PBS) 5ml/10cm plate washing transfectional cell once, scrape in the 5ml same buffer, merge the cell suspension of a plurality of flat boards, centrifugal 10 minutes of 4 ℃ of following 500xg.Reconstituted cell precipitation in ice-cold TEE (25mM TRIS, 5mM EGTA, 5mM EDTA).The suitable equal portions of snap frozen in liquid nitrogen are stored at-70 ℃.After the thawing, cell homogenates, and at 4 ℃, under the 500xg centrifugal 5 minutes, precipitation nuclear and broken cell.Supernatant 4 ℃ with 47, centrifugal 30 minutes of 000xg.Precipitate 1 time with TEE washing film, be suspended in 20mM HEPES again, pH7.4,100mM NaCl, among the 1mM EDTA (test damping fluid), equal portions are also freezing in liquid nitrogen.The film equal portions are stored at-70 ℃.Use that (Rockford, BCA albumen test reagent Illinois) and BSA measure memebrane protein concentration as standard items from Pierce.

[ ³⁵S] GTP γ S is in conjunction with test

Melt the equal portions of cell membrane, homogenate, and dilution goes into to contain 20mM HEPES, pH7.4,100mM NaCl, 10mM MgCl ₂, in the damping fluid of 1mM EDTA (test damping fluid).At first, in 96 hole polypropylene boards (Nunc), be ready to reaction mixture.(20 μ l 10X) or water contrast (20 μ l), be dissolved in 18.2 μ M GDP of test damping fluid (0.11ml, 10 μ M are final), mix with the film (50 microlitres, 10 microgram memebrane proteins) that is suspended in the test damping fluid, place on ice with the aqueous solution of peptide in each hole.Shaking on the platform, room temperature was cultivated part-GDP-film potpourri 20 minutes, placed on ice then.For each sample, add 20 microlitre guanines 5 '-O-(3[ ³⁵S] sulphur) triphosphoric acid ([ ³⁵S] GTP γ S) (600-1200Ci/mmol; From New England Nuclear, Boston, MA) to about 40,000cpm/0.2ml, or ultimate density is 0.1nM.Being added with the flat board of cultivating potpourri (0.2ml/ hole altogether) at room temperature cultivated 45 minutes.Then each reaction mixture equal portions is O.175ml transferred to lavation buffer solution and anticipated in the 96 hole FB MultiScreen filtration dull and stereotyped (Millipore) of (100 microlitres/hole), with MultiScreen vacuum tube (Millipore) vacuum filtration.Use the ice-cold lavation buffer solution/hole of 0.25ml (10mM HEPES, 10mM MgCl then ₂, pH 7.4) and wash film three times, vacuum filtration.After the washing, (25 microlitres/hole, Wallac), seal plate is counted 1 minute/hole in Trilux 1450 Microbeta counters (Wallac) to add the SupermixOpti-phase scintillation solution the last time.As positive control, handle stably express dopamine 2 type (rD with 1mM dopamine (final 100 μ M dopamines) that is dissolved in 0.025% ascorbic acid or carrier ultimate density (0.0025% ascorbic acid) ₂) the Chinese hamster ovary celI film of acceptor.In the presence of the cold GTP γ of 100 μ M S, measure non-specific binding, from sum, deduct.Carrying out each in triplicate handles.

Data analysis

[ ³⁵S] stimulation of inducing of GTP γ S binding partner is expressed as the active multiple in basis that does not add part.Each is handled and to carry out three times, or once in a while twice, be calculated to be mean value ± standard deviation in conjunction with (cpm).With non-linear least square SAS model, y=B _MaxX/ (K _d+ X) dosage of analysis receptor/ligand system relies on curve.(GraphPad Software, Inc.San Diego is CA) with following equation y=bottom+(top-bottom)/(1+10 with Prism ^LogEC ₅₀ ^-X) analyze other dose dependent curve.

The result

Whether further at first, we have selected GTP γ S test as functional trial, because the activation of the GTP γ S that activator drives has reflected the early reaction in the DmGPCR activation cascade, no matter and the activated channel of the various downstream signal transfer reactions of activation.This it seems that to have the activation of orphan DmGPCR of unknown function and unknown signaling pipeline for assessment particularly useful.Filter with 96 hole MultiScreen G/FB filters and MultiScreen vacuum tube (Millipore), carry out GTP γ S with the film of the Chinese hamster ovary celI preparation of the DNA transient transfection of coding fruit bat GPCR and test.Because the GPCR with the Gq family coupling of G albumen can not be well discerned in known GTP γ S test, uses the Ca based on the FLIPR reading ²⁺The be encoded orphan GPCR of Gq-coupling in the Chinese hamster ovary celI of DNA transient transfection of fruit bat GPCR of migration test assessment.

Use GTP γ S test, found that DmGPCR1 (PnuFlyPep34651) is by two kinds of fruit bat NPF-sample peptide: AQRSPSLRLRF-NH ₂(SEQ ID NO:186) and PIRSPSLRLRF-NH ₂(SEQ ID NO:187) activates, as the EC of the about 2.5nM that measures ₅₀What value reflected is such.Use DPKQDFMRF-NH ₂(SEQ ID NO:26) and PDNFMRF-NH ₂(SEQ ID NO:27) activates the EC that causes GTP γ S reaction ₅₀Scope is at 370nM-500nM.As Nambu etc. (Neuron, 1988,1, reported that 55-61) these two peptides and 9 other FaRP are encoded by same precursor-gene.Though the relatively poor (EC of effect ₅₀Scope is 5-10 μ M), other FaRP and other also stimulate the neuropeptide of GTP γ S combination to comprise following peptide: TDVDHVFLRF-NH ₂(SEQ IDNO:25), TPAEDFMRF-NH ₂(SEQ ID NO:28), SLKQDFMHF-NH ₂(SEQ ID NO:29), SVKQDFMHF-NH ₂(SEQ ID NO:30), AAMDRY-NH ₂(SEQ ID NO:31), and SVQDNFMHF-NH ₂(SEQ ID NO:32).In addition, the FLIPR test identifies Colorado potato bug peptide ARGPQLRLRF-NH ₂(SEQ ID NO:33) is complementary EC with the DmGPCR1 acceptor ₅₀Be 100-200nM.Our data show that Dmgpcr1 should be categorized as short neuropeptide F acceptor, because it can be by two short NPF peptides, SEQ ID NO:186 and SEQ ID NO:187 activate strongly.

Shown in GTP γ S reaction, DmGPCR4 (PnuFlyPep 67393) is by the fruit bat allatostatin, fruit bat inhibin-3 (SRPYSFGL-NH ₂(SEQ ID NO:165)) to hang down the EC of nanomole scope ₅₀, and various cockroach (Diplotera punctata) allatostatin, i.e. GDGRLYAFGL-NH ₂(SEQ ID NO:34), DRLYSFGL-NH ₂(SEQ ID NO:35), APSGAQRLYGFGL-NH ₂(SEQ ID NO:36), and GGSLYSFGL-NH ₂(SEQ ID NO:37) (EC ₅₀The about 20-280nM of scope) activates.When testing with FLIPR with 10 μ M, same peptide causes very strong calcium signal.Recently, Lenz etc. (on seeing) have cloned DmGPCR4, and are categorized into the allatostatin acceptor (DARII) that second class is inferred.Yet, receptor activation is not had the report of any materia medica data so far.As far as our knowledge goes, this is that various allatostatins activate this receptor experimental evidence the earliest really.

Shown in GTP γ S reaction, when transient expression in the CHO-10001A cell, DmGPCR5 (GenBank accession number AX128628) can be by fruit bat tachykinin (DTK), i.e. DTK-1 (APTSSFIGMR-NH ₂) (SEQ ID NO:169), Met8-DTK-2 (APLAFYGMR-NH ₂) (SEQID NO:170), DTK-2 (APLAFYGLR-NH ₂) (SEQ ID NO:171), DTK-3 (APTGFTGMR-NH ₂) (SEQ ID NO:172), DTK-4 (APVNSFVGMR-NH ₂) (SEQ ID NO:173), and DTK-5 (APNGFLGMR-NH ₂) (SEQ ID NO:174) activation.Rely in the test at dosage, DTK-1, Met8-DTK-2, DTK-3 and DTK-4 are with the EC of 250-500nM scope ₅₀Stimulate GTP γ S combination, maximum stimulation is about 1.5 times more than the baseline values.DTK-2 and DTK-4 are by the EC in its low micro-molar range ₅₀Judge, a little less than.In calcium migration test (FLIPR), DmGPCR5 shows that identical DTK is had EC in the 1-20nM scope ₅₀Ca ^{+ 2}Reaction.In addition, test DTK-5 in the test of cAMP (based on reporter), DTK-2 and Met8-DTK-2 are with EC ₅₀The dosage dependence form that is respectively 197nM, 1.06 μ M and 583nM stimulates cAMP to discharge.These data show, DmGPCR5 and Gs (cAMP) and Gq (Ca ^{+ 2}The signal pipeline coupling of)-mediation, the signal pipeline that it and vertebrate tachykinin receptor are reported is similar.

DmGPCR6a (M811490) by Li etc. (J.Biol.Chem., 1992,267,9-12) be reported as pyy receptor.With GTP γ S test, under 5 μ M concentration, tested the peptide that table 7 is listed, they stimulate GTP γ S to combine (the above 1.7-4 of baseline is doubly) with the Chinese hamster ovary celI film of the DNA transfection of encoding D mGPCR6a.It should be noted that except a collection of insect and nematode peptide of activating this receptor, also finder NPFF (FLFQPQRF-NH ₂(SEQID NO:59)) also be the part (5 μ M NPFF make GTP γ S in conjunction with having improved 4 times) of DmGPCR6.

Dmgcr6aL and Dmgpcr6bL are two kinds of splice variants of DmGPCR6a (M811490).Latter Li etc. (J.Biol.Chem., 1992,267,9-12) be reported as pyy receptor.We claim that DmGPCR6aL and DmGPCR6bL are RF-acid amides acceptors, have Arg-Phe-NH because they only are identified in the C end ₂(Rfa) peptide of sequence.These DmGPCR not the peptide of " identification " C-terminal different with the Rfa sequence (for example SFa, QFa, YFa, RLa, DWa, RPa, HFa, LQa, SNa etc.).In calcium migration test (FLIPR), Dmgpcr6aL and Dmgpcr6bL show the very strong Ca in FaRP storehouse with 10 μ M test ^{+ 2}Reaction.Hereinafter the sequence that shows of the table 7 active FaRP that represented all to identify belongs to different plant species, comprises fruit bat, nematode, roundworm (A.suum), mollusc, P.redivivus (a kind of nematode), fluke, lobster, people and leech.The sole exception of C-end " RF acid amides rule " is peptide pGluDRDYRPLQF-NH2 (SEQ ID NO:120), and its C end contains Gln-Phe-NH ₂(QFa) sequence.What is interesting is that Dmgpcr6aL and Dmgpcr6bL discern NPFF (FLFQPQRF-NH ₂(SEQ ID NO:152)), a kind of mammalian-derived peptides (p-Glu or pQ represent pyroglutamic acid in the attention The above results) that has RF acid amides sequence at the C-end.

Shown in RLIPR analyzes, the DmGPCR7 of transient expression in the CHO-10001A cell (GenBank accession number AX128636) is activated by leucokinin (LK) and related peptides, be LK-I (DPAFNSWGa) (SEQID NO:175), LK-V (GSGFSSWGa) (SEQ ID NO:176), LK-VI (pGlu-SSFHSWGa) (SEQ ID NO:177), LK-VIII (GSAFYSWGa) (SEQ ID NO:178), culex kassinin kinin (NPFHSWGa) (SEQ ID NO:179), mollusc lymphocyte kassinin kinin sample peptide, be snail kassinin kinin (PSFHSWSa) (SEQ ID NO:180), with fruit bat leucokinin sample peptide DLK-1 (NSVVLGKKQRFHSWGa) (SEQ ID NO:181), DLK-2 (pGlu-RFHSWGa) (SEQ IDNO:182) and DLK-2A (QRFHSWGa) (SEQ ID NO:183).The LK peptide that DmGPCR7 can be had the terminal tetrapeptide array HSWGa of common C-activates.Handle (comprising DLK-1, DLK-2, DLK-2a, LK-VI and culex kassinin kinin) with this group peptide, cause very strong cellular calcium to discharge (EC ₅₀For picomole below nanomole).On the contrary, other locust LK and mollusc LK (SEQ IDNO:180) with terminal S/NSWGa of C-(LK-1, LK-V) shows lower ability (EC ₅₀Be 15-30nM), have LK-VIII the most weak (EC of ability in this series of YSWGaC-end sequence ₅₀Be the 100-200nM scope).Be indicated as G _Q/11-coupled receptor reacts with the GTP γ S that can not detect in the film of DmGPCR7/CHO cell preparation these peptides.Therefore, to be accredited as the leucokinin acceptor that the calcium signal transmits (more may be G to DmGPCR7 _Q/11-coupling), mate as cognate ligand with the fruit bat leucokinin.

Shown in GTP γ S reaction, the DmGPCR of transient expression in the CHO-10001A cell (GenBank accession number AX128639) is by hawkmoth (Manduca sexta) allatostatin-C (AST-C or Manse-C)

(SEQ ID NO:184) or fruit bat inhibin-C (DST-C) are also referred to as platform line peptide (FLT)

(SEQ ID NO:185).In conjunction with in testing, detect AST-C and DST-C reaction (EC efficiently at dose response GTP γ S- ₅₀Be low nanomole scope).Use the pertussis toxin pretreatment cell, eliminated these activity fully, the indication receptor activation is relevant with Gi/Go.In direct calcium migration test (FLIPR), when attacking with AST-C or DST-C, DmGPCR8 does not show any activity.Yet the strong calcium that detects in the CHO-10001A cell with DmGPCR8 and chimeric G-Protein G qi5 or Gqo5 cotransfection DST-C discharges active (EC ₅₀About 30nM).On the other hand, with the relatively poor (EC of Gqz5 coupling effect ₅₀244nm), in cell, do not observe the calcium migration with DmGPCR8 and Gqs5 cotransfection.These results show that DmGPCR8 is the inhibition acceptor in the Chinese hamster ovary celI, it preferably with Gi/Go type G albumen coupling.Representational result understands is accredited as DmGPCR8 the DST-C/FLT acceptor.

Very strong based on its signal in the calcium migration test, DmGPCR9 with FDDY (SO ₃H) GHLRF-NH ₂(SEQ ID NO:157) mates (EC ₅₀Be low nanomole scope).Film with the preparation of the DNA transfection CHO cell of encoding D mGPCR9 detects less than the GTP γ S reaction to this peptide, shows DmGPCR9 most probable and the coupling of Gq signal pipeline.FDDY (SO ₃H) GHLRF-NH ₂(SEQ IDNO:157) represents natural fruit bat sulfanilamide (SN) kassinin kinin-1 (DSK-1), FDDY (SO ₃H) GHMRF-NH ₂Met7 → Leu7 the analog of (SEQ ID NO:159).Therefore, we think that the DmGPCR9 acceptor is the sulfanilamide (SN) kinin receptor.Because FDDYGHLRF-NH ₂(SEQ ID NO:158) is as FDDY (SO ₃H) GHLRF-NH ₂The non-sulfanilamide (SN) homologue of (SEQ IDNO:157), show very weak calcium signal when under 10 μ M, detecting, and the FARP of other 117 kinds of tests and relevant peptide all do not show any activity in the FLIPR of DmGPCR acceptor or GTP γ S test, thinks that this coupling is very specific.

Hereinafter table 7 has shown the coupling of part and its associated receptor.

???? GPCR	? SEQ?ID?NO	The peptide matching sequence
			????dmgpcr1	?SEQ?ID?NO：186	????AQRSPSLRLRF-NH 2
?SEQ?ID?NO：187	????PIRSPSLRLRF-NH 2
		?SEQ?ID?NO：25		????TDVDHVFLRF-NH 2
?SEQ?ID?NO：26	????DPKQDFMRF-NH 2
		?SEQ?ID?NO：27		????PDNFMRF-NH 2
?SEQ?ID?NO：28	????TPAEDFMRF-NH 2
		?SEQ?ID?NO：29		????SLKQDFMHF-NH 2
?SEQ?ID?NO：30	????SVKQDFMHF-NH 2
		?SEQ?ID?NO：31		????AAMDRY-NH 2
?SEQ?ID?NO：32	????SVQDNFMHF-NH 2
		?SEQ?ID?NO：33		????ARGPQLRLRF-NH 2
????dmgpcr4	?SEQ?ID?NO：34				????GDGRLYAFGL-NH 2
		?SEQ?ID?NO：35	????DRLYSFGL-NH 2
	?SEQ?ID?NO：36			????APSGAQRLYGFGL-NH 2
		?SEQ?ID?NO：37	????GGSLYSFGL-NH 2
	????dmgpcr6 ????(6a)			?SEQ?ID?NO：38	????FIRF-NH 2
?SEQ?ID?NO：39		????KNEFIRF-NH 2
			?SEQ?ID?NO：40	????FMRF-NH 2
?SEQ?ID?NO：41		????KSAFMRF-NH 2
			?SEQ?ID?NO：42	????KPNFLRF-NH 2
?SEQ?ID?NO：43		????FLRF-NH 2
			?SEQ?ID?NO：44	????YLRF-NH 2
?SEQ?ID?NO：45		????KPNFLRY-NH 2
			?SEQ?ID?NO：46	????TNRNFLRF-NH 2
?SEQ?ID?NO：47		????RNKFEFIRF-NH 2
			?SEQ?ID?NO：48	????AGPRFIRF-NH 2
?SEQ?ID?NO：49		????GLGPRPLRF-NH 2
			?SEQ?ID?NO：50	????IL-Nle-RF-NH 2
?SEQ?ID?NO：51		????AGAKFIRF-NH 2
			?SEQ?ID?NO：52	????APKPKFIRF-NH 2
?SEQ?ID?NO：53		????KSAFVLRF-NH 2
			?SEQ?ID?NO：54	????TKFQDFLRF-NH 2
?SEQ?ID?NO：55		????SAEPFGTMRF-NH 2
			?SEQ?ID?NO：56	????ASEDALFGTMRF-NH 2
?SEQ?ID?NO：57		????SADDSAPFGTMRF-NH 2
			?SEQ?ID?NO：58	????EDGNAPFGTMRF-NH 2
?SEQ?ID?NO：59		????FLFQPQRF-NH 2
			Dmgpcr6 6aL and 6bL	?SEQ?ID?NO：60	????SADPNFLRF-NH 2
?SEQ?ID?NO：61	????SQPNFLRF-NH 2
		?SEQ?ID?NO：62		????ASGDPNFLRF-NH 2
?SEQ?ID?NO：63	????SDPNFLRF-NH 2
		?SEQ?ID?NO：64		????AAADPNFLRF-NH 2
?SEQ?ID?NO：65	????PNFLRF-NH 2
		?SEQ?ID?NO：66		????KPNFLRF-NH 2
?SEQ?ID?NO：67	????AGSDPNFLRF-NH 2
		?SEQ?ID?NO：68		????KPNFLRY-NH 2

	SEQ?ID?NO：69	????SPREPIRF-NH 2
			SEQ?ID?NO：70	????LRGEPIRF-NH 2
	SEQ?ID?NO：71	????SPLGTMRF-NH 2
			SEQ?ID?NO：72	????EAEEPLGTMRF-NH 2
	SEQ?ID?NO：73	????ASEDALFGTMRF-NH 2
			SEQ?ID?NO：74	????EDGNAPFGTMRF-NH 2
	SEQ?ID?NO：75	????SAEPFGTMRF-NH 2
			SEQ?ID?NO：76	????SADDSAPFGTMRF-NH 2
	SEQ?ID?NO：77	????KPTFIRF-NH 2
			SEQ?ID?NO：78	????ASPSFIRF-NH 2
	SEQ?ID?NO：79	????GAKFIRF-NH 2
			SEQ?ID?NO：80	????AGAKFIRF-NH 2
	SEQ?ID?NO：81	????APKPKFIRF-NH 2
			SEQ?ID?NO：82	????KSAYMRF-NH 2
	SEQ?ID?NO：83	????SPMQRSSMVRF-NH 2
			SEQ?ID?NO：84	????SPMERSAMVRF-NH 2
	SEQ?ID?NO：85	????SPMDRSKMVRF-NH 2
			SEQ?ID?NO：86	????KNEFIRF-NH 2
	SEQ?ID?NO：87	????KPSFVRF-NH 2
			SEQ?ID?NO：88	????pQPKARSGYIRF-NH 2
	SEQ?ID?NO：89	????AMRNALVRF-NH 2
			SEQ?ID?NO：90	????ASGGMRNALVRF-NH 2
	SEQ?ID?NO：91	????NGAPQPFVRF-NH 2
			SEQ?ID?NO：92	????RNKFEFIRF-NH 2
	SEQ?ID?NO：93	????SDRPTRAMDSPLIRF-NH 2
			SEQ?ID?NO：94	????AADGAPLIRF-NH 2
	SEQ?ID?NO：95	????APEASPFIRF-NH 2
			SEQ?ID?NO：96	????ASPSAPLIRF-NH 2
	SEQ?ID?NO：97	????SPSAVPLIRF-NH 2
			SEQ?ID?NO：98	????ASSAPLIRF-NH 2
	SEQ?ID?NO：99	????KHEYLRF-NH 2
			SEQ?ID?NO：100	????SLLDYRF-NH 2
	SEQ?ID?NO：101	????EIVFHQISPIFFRF-NH 2
			SEQ?ID?NO：102	????GGPQGPLRF-NH 2
	SEQ?ID?NO：103	????GPSGPLRF-NH 2
			SEQ?ID?NO：104	????AQTFVRF-NH 2
	SEQ?ID?NO：105	????GQTFVRF-NH 2
			SEQ?ID?NO：106	????KSAFVRF-NH 2
	SEQ?ID?NO：107	????KSQYIRF-NH 2
			SEQ?ID?NO：108	????DVPGVLRF-NH 2
	SEQ?ID?NO：109	????KSVPGVLRF-NH 2
			SEQ?ID?NO：110	????SEVPGVLRF-NH 2
	SEQ?ID?NO：111	????SVPGVLRF-NH 2
			SEQ?ID?NO：112	????DFDGAMPGVLRF-NH 2
	SEQ?ID?NO：113	????EIPGVLRF-NH 2
			SEQ?ID?NO：114	????WANQVRF-NH 2
	SEQ?ID?NO：115	????ASWASSVRF-NH 2

	?SEQ?ID?NO：116		????AMMRF-NH 2
				?SEQ?ID?NO：117		????GLGPRPLRF-NH 2
	?SEQ?ID?NO：118		????SPSAKWMRF-NH 2
				?SEQ?ID?NO：119		????TKFQDFLRF-NH 2
	?SEQ?ID?NO：120		????pQDRDYRPLQF-NH 2
				?SEQ?ID?NO：121		????FIRF-NH 2
	?SEQ?ID?NO：122		????AVPGVLRF-NH 2
				?SEQ?ID?NO：123		????GDVPGVLRF-NH 2
	?SEQ?ID?NO：124		????SDIGISEPNFLRF-NH 2
				?SEQ?ID?NO：125		????SGKPTFIRF-NH 2
	?SEQ?ID?NO：126		????AEGLSSPLIRF-NH 2
				?SEQ?ID?NO：127		????FDRDFMRF-NH 2
	?SEQ?ID?NO：128		????AGPRFIRF-NH 2
				?SEQ?ID?NO：129		????GMPGVLRF-NH 2
	?SEQ?ID?NO：130		????IL-Nle-RF-NH 2
				?SEQ?ID?NO：131		????LQPNFLRF-NH 2
	?SEQ?ID?NO：132		????KPNFIRF-NH 2
				?SEQ?ID?NO：133		????FMRF-NH 2
	?SEQ?ID?NO：134		????FLRF-NH 2
				?SEQ?ID?NO：135		????YIRF-NH 2
	?SEQ?ID?NO：136		????GNSFLRF-NH 2
				?SEQ?ID?NO：137		????DPSFLRF-NH 2
	?SEQ?ID?NO：138		????pQDFMRF-NH 2
				?SEQ?ID?NO：139		????KPNQDFMRF-NH 2
	?SEQ?ID?NO：140		????TDVDHVFLRF-NH 2
				?SEQ?ID?NO：141		????AAMDRY-NH 2
	?SEQ?ID?NO：142		????SPKQDFMRF-NH 2
				?SEQ?ID?NO：143		????PDNFMRF-NH 2
	?SEQ?ID?NO：144		????DPKQDFMRF-NH 2
				?SEQ?ID?NO：145		????TPAEDFMRF-NH 2
	?SEQ?ID?NO：146		????SDNFMRF-NH 2
				?SEQ?ID?NO：147		????YLRF-NH 2
	?SEQ?ID?NO：148		????SDRNFLRF-NH 2
				?SEQ?ID?NO：149		????TNRNFLRF-NH 2
	?SEQ?ID?NO：150		????PDVDHVFLRF-NH 2
				?SEQ?ID?NO：151		????pQDVDHVFLRF-NH 2
	?SEQ?ID?NO：152		????FLFQPQRF-NH 2
				?SEQ?ID?NO：153		????ARGPQLRLRF-NH 2
	?SEQ?ID?NO：154		????FDDY(SO3H)GHLRF-NH 2
				?SEQ?ID?NO：155		????FDDYGHLRF-NH 2
	?SEQ?ID?NO：156		????MDSNFIRF-NH 2
				????dmgpcr9	?SEQ?ID?NO：157		????FDDY(SO3H)GHLRF-NH 2
????dmgpcr5	?SEQ?ID?NO：169		????APTSSFIGMR-NH 2
					?SEQ?ID?NO：170		????APLAFYGMR-NH 2
	?SEQ?ID?NO：171		????APLAFYGLR-NH 2
					?SEQ?ID?NO：172		????APTGFTGMR-NH 2
	?SEQ?ID?NO：173		????APVNSFVGMR-NH 2
					?SEQ?ID?NO：174		????APNGFLGMR-NH 2
????dmgpcr7	?SEQ?ID?NO：175		????DPAFNSWG-NH 2

Embodiment 10: competitive trials

The preparation of list-iodate peptide

By typical toluene-sodium-sulfonchloramide method iodate peptide.In the 2ml vial, add the 1mM peptide aqueous solution of 10 microlitres, sodium phosphate buffer, the 1.0mCi[of 10 microlitre 0.1M (pH7.99) ¹²⁵I] the 2mg/ml toluene-sodium-sulfonchloramide solution (in phosphate buffer) of sodium iodide and 5 microlitres.The potpourri vortex vibrates 60 seconds, adds the phosphate-buffered liquor of the sodium metabisulfite of 25 microlitre 5mg/ml.Potpourri carries out HPLC then, it is expelled to Vydac C18 (on 0.45 * 15cm) post, carries out gradient separations.Used gradient is to be 0 o'clock 70%A and 30%B in the time, during by 25 minutes be 20%A and 80%B (aqueous solution of A=0.1M ammonium acetate, the aqueous solution 40% of B=0.1M ammonium acetate: CH3CN60%, v/v).Flow velocity is 1.0ml/ minute.From t=8 to t=20 minute, sample collection is gone into 0.25ml capture buffer liquid (the 0.1M sodium phosphate buffer contains 0.5% bovine serum albumin, 0.1%Triton X100 and 0.05%Tween 20) every 30 seconds.Single iodate peptide usually in the time of t=11 minute by wash-out, productive rate is about 0.75ml/100 μ Ci.

In conjunction with test

96 used orifice plates are Millipore Multiscreen  filter (the opaque 1.0 μ M glass fibre Type Bs of FB, catalog number (Cat.No.) MAFBNOB50).Unite and use Millipore Multiscreen  solvent and awarding property pipe (catalog number (Cat.No.) MAVMO960R) and 96 orifice plates, filter analysis thing during end.Every hole portion contains 5 micrograms of protein (above-mentioned prepared product) in 100 microlitre volumes, each test group comprises two equal portions.For each test compounds, only use [125I] peptide to carry out (being used for total binding) for one group, one group with the test compounds of 1 μ M (or as specify) concentration and [ ¹²⁵I] peptide (being used for non-specific binding) operation.Every part of order that adds reagent is: test damping fluid (20mM HEPES, 10mMMgCl ₂, 1% bovine serum albumin, pH7.4), test compounds (preparation test damping fluid in), [ ¹²⁵I] peptide (in the test damping fluid) and film suspension (in the test damping fluid).Add the film suspension and caused association reaction, carried out 30 minutes in room temperature (22 ℃).After cultivating in 30 minutes, Jiang Geban places on the strainer tube, adds vacuum, makes liquid pass through filter membrane (abandoning), on the filter membrane of protein capture in each hole.For washing, discharge vacuum, in each hole, add 200 μ l test damping fluid, and then add vacuum.Washing repeats twice (altogether to each equal portions washing 3 times) again.After the washing, remove the beneath vinyl cover in each hole, flat board is placed the Microbeta  scintillation counting box (catalog number (Cat.No.) 1450-105) of bottom sealing.In each hole, add 25 microlitre scintillation solutions, plate was placed on the rotation jolting device 80rpm 1 hour, then standing over night.Next day, flat board is counted on Microbeta  scintillation counter.From average total binding, deduct average non-specific binding, obtain standard (peptide amide) and unknown specificity combination.

It will be understood to those of skill in the art that and can carry out many changes and modification the embodiment of the invention described above, and without prejudice to spirit of the present invention.Should understand all changes all within the scope of the invention.

The whole disclosure of the publication that all this paper quote all is incorporated herein for your guidance.

Sequence table

＜110〉(the PHARMACIA ﹠amp of Pharmacia ﹠ Upjohn Company LLC; UPJOHN COMPANY LLC)

＜120〉fruit bat g protein coupled receptor, nucleic acid and correlation technique

<130>PHRM0002-103

<150>US10/283,423

<151>2002-10-30

<150>US?09/693,746

<151>2000-10-20

<150>US?09/425,676

<151>1999-10-22

<160>187

<170>PatentIn?version?3.2

<210>1

<211>1803

<212>DNA

＜213〉fruit bat (D.melanogaster)

<400>1

atggccaact?taagctggct?gagcaccatc?accaccacct?cctcctccat?cagcaccagc?????60

cagctgccat?tggtcagcac?aaccaactgg?agcctaacgt?cgccgggaac?tactagcgct????120

atcttggcgg?atgtggctgc?atcggatgag?gataggagcg?gcgggatcat?tcacaaccag????180

ttcgtgcaaa?tcttcttcta?cgtcctgtac?gccacggtct?ttgtcctggg?tgtcttcgga????240

aatgtcctgg?tttgctacgt?agttctgagg?aatcgggcca?tgcagactgt?gaccaatata????300

ttcatcacga?atctggccct?gtcggacata?ttgctctgcg?tcctggcggt?gccatttact????360

ccgctttaca?cgttcatggg?tcgctgggcc?ttcggcagga?gtctgtgcca?tctggtgtcc????420

tttgcccagg?gatgcagcat?ctacatatcc?acgctgaccc?tcacctcgat?tgccatcgat????480

cggtacttcg?ttatcatata?ccccttccat?ccgcgcatga?agctctccac?ctgcatcggg????540

atcatagtga?gcatctgggt?gatagccctg?ctggccaccg?ttccctacgg?catgtacatg????600

aagatgacca?acgagctggt?gaacggaacg?cagacaggca?acgagaccct?ggtggaggcc????660

actctaatgc?taaacggaag?ctttgtggcc?cagggatcag?gattcatcga?ggcgccggac????720

tctacctcgg?ccacccaggc?ctatatgcag?gtgatgaccg?ccggatcaac?gggaccggag????780

atgccctatg?tgcgggtgta?ctgcgaggag?aactggccat?cggagcagta?ccggaaggtg????840

ttcggtgcca?tcacaaccac?tctgcagttt?gtgctgccct?tcttcatcat?ctcgatttgc????900

tacgtgtgga?tatcggtgaa?gctaaaccag?cgggccaggg?ccaagccggg?atcgaaatcc????960

tcgagacggg?aggaggcgga?tcgggatcgc?aagaagcgca?ccaaccgcat?gctcatcgcc???1020

atggtggcgg?tattcggact?cagctggctg?cccatcaatg?tggtcaacat?attcgatgac???1080

ttcgatgaca?agtccaacga?gtggcgcttc?tacatcctat?tcttctttgt?ggcccactct???1140

attgccatga?gctccacctg?ctacaatccc?ttcctgtacg?cctggctgaa?cgagaacttc???1200

cgcaaggagt?tcaagcacgt?gctgccctgc?tttaatccct?cgaacaacaa?catcatcaac???1260

atcaccaggg?gctataatcg?gagtgatcgg?aacacctgtg?gtccgcgact?gcatcatggc???1320

aagggggatg?gtggcatggg?cggtggcagt?ctggacgccg?acgaccagga?cgagaacggc???1380

atcacccagg?agacctgtct?gcccaaggag?aagctgctga?ttatccccag?ggagccgact???1440

tacggcaatg?gcacgggtgc?cgtgtcgcca?atccttagcg?ggcgcggcat?taacgccgcc???1500

ctggtgcacg?gtggcgacca?tcagatgcac?cagctgcagc?cgtcacacca?tcaacaggtg???1560

gagctgacga?ggcgaatccg?ccggcggaca?gacgagacgg?acggggatta?cctggactcc???1620

ggcgacgagc?agaccgtgga?ggtgcgcttc?agcgagacgc?cgttcgtcag?cacggataat???1680

accaccggga?tcagcattct?ggagacgagt?acgagtcact?gccaggactc?ggatgtgatg???1740

gtcgagctgg?gcgaggcaat?cggcgccggt?ggtggggcag?agctggggag?gcgaatcaac???1800

tga?????????????????????????????????????????????????????????????????1803

<210>2

<211>600

<212>PRT

＜213〉fruit bat (D.melanogaster)

<400>2

Met?Ala?Asn?Leu?Ser?Trp?Leu?Ser?Thr?Ile?Thr?Thr?Thr?Ser?Ser?Ser

1???????????????5???????????????????10??????????????????15

Ile?Ser?Thr?Ser?Gln?Leu?Pro?Leu?Val?Ser?Thr?Thr?Asn?Trp?Ser?Leu

20??????????????????25??????????????????30

Thr?Ser?Pro?Gly?Thr?Thr?Ser?Ala?Ile?Leu?Ala?Asp?Val?Ala?Ala?Ser

35??????????????????40??????????????????45

Asp?Glu?Asp?Arg?Ser?Gly?Gly?Ile?Ile?His?Asn?Gln?Phe?Val?Gln?Ile

50??????????????????55??????????????????60

Phe?Phe?Tyr?Val?Leu?Tyr?Ala?Thr?Val?Phe?Val?Leu?Gly?Val?Phe?Gly

65??????????????????70??????????????????75??????????????????80

Asn?Val?Leu?Val?Cys?Tyr?Val?Val?Leu?Arg?Asn?Arg?Ala?Met?Gln?Thr

85??????????????????90??????????????????95

Val?Thr?Asn?Ile?Phe?Ile?Thr?Asn?Leu?Ala?Leu?Ser?Asp?Ile?Leu?Leu

100?????????????????105?????????????????110

Cys?Val?Leu?Ala?Val?Pro?Phe?Thr?Pro?Leu?Tyr?Thr?Phe?Met?Gly?Arg

115?????????????????120?????????????????125

Trp?Ala?Phe?Gly?Arg?Ser?Leu?Cys?His?Leu?Val?Ser?Phe?Ala?Gln?Gly

130?????????????????135?????????????????140

Cys?Ser?Ile?Tyr?Ile?Ser?Thr?Leu?Thr?Leu?Thr?Ser?Ile?Ala?Ile?Asp

145?????????????????150?????????????????155?????????????????160

Arg?Tyr?Phe?Val?Ile?Ile?Tyr?Pro?Phe?His?Pro?Arg?Met?Lys?Leu?Ser

165?????????????????170?????????????????175

Thr?Cys?Ile?Gly?Ile?Ile?Val?Ser?Ile?Trp?Val?Ile?Ala?Leu?Leu?Ala

180?????????????????185?????????????????190

Thr?Val?Pro?Tyr?Gly?Met?Tyr?Met?Lys?Met?Thr?Asn?Glu?Leu?Val?Asn

195?????????????????200?????????????????205

Gly?Thr?Gln?Thr?Gly?Asn?Glu?Thr?Leu?Val?Glu?Ala?Thr?Leu?Met?Leu

210?????????????????215?????????????????220

Asn?Gly?Ser?Phe?Val?Ala?Gln?Gly?Ser?Gly?Phe?Ile?Glu?Ala?Pro?Asp

225?????????????????230?????????????????235?????????????????240

Ser?Thr?Ser?Ala?Thr?Gln?Ala?Tyr?Met?Gln?Val?Met?Thr?Ala?Gly?Ser

245?????????????????250?????????????????255

Thr?Gly?Pro?Glu?Met?Pro?Tyr?Val?Arg?Val?Tyr?Cys?Glu?Glu?Asn?Trp

260?????????????????265?????????????????270

Pro?Ser?Glu?Gln?Tyr?Arg?Lys?Val?Phe?Gly?Ala?Ile?Thr?Thr?Thr?Leu

275?????????????????280?????????????????285

Gln?Phe?Val?Leu?Pro?Phe?Phe?Ile?Ile?Ser?Ile?Cys?Tyr?Val?Trp?Ile

290?????????????????295?????????????????300

Ser?Val?Lys?Leu?Asn?Gln?Arg?Ala?Arg?Ala?Lys?Pro?Gly?Ser?Lys?Ser

305?????????????????310?????????????????315?????????????????320

Ser?Arg?Arg?Glu?Glu?Ala?Asp?Arg?Asp?Arg?Lys?Lys?Arg?Thr?Asn?Arg

325?????????????????330?????????????????335

Met?Leu?Ile?Ala?Met?Val?Ala?Val?Phe?Gly?Leu?Ser?Trp?Leu?Pro?Ile

340?????????????????345?????????????????350

Asn?Val?Val?Asn?lle?Phe?Asp?Asp?Phe?Asp?Asp?Lys?Ser?Asn?Glu?Trp

355?????????????????360?????????????????365

Arg?Phe?Tyr?Ile?Leu?Phe?Phe?Phe?Val?Ala?His?Ser?Ile?Ala?Met?Ser

370?????????????????375?????????????????380

Ser?Thr?Cys?Tyr?Asn?Pro?Phe?Leu?Tyr?Ala?Trp?Leu?Asn?Glu?Asn?Phe

385?????????????????390?????????????????395?????????????????400

Arg?Lys?Glu?Phe?Lys?His?Val?Leu?Pro?Cys?Phe?Asn?Pro?Ser?Asn?Asn

405?????????????????410?????????????????415

Asn?Ile?Ile?Asn?Ile?Thr?Arg?Gly?Tyr?Asn?Arg?Ser?Asp?Arg?Asn?Thr

420?????????????????425?????????????????430

Cys?Gly?Pro?Arg?Leu?His?His?Gly?Lys?Gly?Asp?Gly?Gly?Met?Gly?Gly

435?????????????????440?????????????????445

Gly?Ser?Leu?Asp?Ala?Asp?Asp?Gln?Asp?Glu?Asn?Gly?Ile?Thr?Gln?Glu

450?????????????????455?????????????????460

Thr?Cys?Leu?Pro?Lys?Glu?Lys?Leu?Leu?Ile?Ile?Pro?Arg?Glu?Pro?Thr

465?????????????????470?????????????????475????????????????480

Tyr?Gly?Asn?Gly?Thr?Gly?Ala?Val?Ser?Pro?Ile?Leu?Ser?Gly?Arg?Gly

485?????????????????490?????????????????495

Ile?Asn?Ala?Ala?Leu?Val?His?Gly?Gly?Asp?His?Gln?Met?His?Gln?Leu

500?????????????????505?????????????????510

Gln?Pro?Ser?His?His?Gln?Gln?Val?Glu?Leu?Thr?Arg?Arg?Ile?Arg?Arg

515?????????????????520?????????????????525

Arg?Thr?Asp?Glu?Thr?Asp?Gly?Asp?Tyr?Leu?Asp?Ser?Gly?Asp?Glu?Gln

530?????????????????535?????????????????540

Thr?Val?Glu?Val?Arg?Phe?Ser?Glu?Thr?Pro?Phe?Val?Ser?Thr?Asp?Asn

545?????????????????550?????????????????555?????????????????560

Thr?Thr?Gly?Ile?Ser?Ile?Leu?Glu?Thr?Ser?Thr?Ser?His?Cys?Gln?Asp

565?????????????????570?????????????????575

Ser?Asp?Val?Met?Val?Glu?Leu?Gly?Glu?Ala?Ile?Gly?Ala?Gly?Gly?Gly

580?????????????????585?????????????????590

Ala?Glu?Leu?Gly?Arg?Arg?Ile?Asn

595?????????????????600

<210>3

<211>1445

<212>DNA

＜213〉fruit bat (D.melanogaster)

<400>3

atgaatcaga?cggagcccgc?ccagctggca?gatggggagc?atctgagtgg?atacgccagc?????60

agcagcaaca?gcgtgcgcta?tctggacgac?cggcatccgc?tggactacct?tgacctgggc????120

acggtgcacg?ccctcaacac?cactgccatc?aacacctcgg?atctgaatga?gactgggagc????180

aggccgctgg?acccggtgct?tatcgatagg?ttcctgagca?acagggcggt?ggacagcccc????240

tggtaccaca?tgctcatcag?catgtacggc?gtgctaatcg?tcttcggcgc?cctaggcaac????300

accctggttg?ttatagccgt?catccggaag?cccatcatgc?gcactgctcg?caatctgttc????360

atcctcaacc?tggccatatc?ggacctactt?ttatgcctag?tcaccatgcc?gctgaccttg????420

atggagatcc?tgtccaagta?ctggccctac?ggctcctgct?ccatcctgtg?caaaacgatt????480

gccatgctgc?aggcactttg?tattttcgtg?tcgacaatat?ccataacggc?cattgccttc????540

gacagatatc?aggtgatcgt?gtaccccacg?cgggacagcc?tgcagttcgt?gggcgcggtg????600

acgatcctgg?cggggatctg?ggcactggca?ctgctgctgg?cctcgccgct?gttcgtctac????660

aaggagctga?tcaacacaga?cacgccggca?ctcctgcagc?agatcggcct?gcaggacacg????720

atcccgtact?gcattgagga?ctggccaagt?cgcaacgggc?gcttctacta?ctcgatcttc????780

tcgctgtgcg?tacaatacct?ggtgcccatc?ctgatcgtct?cggtggcata?cttcgggatc????840

tacaacaagc?tgaagagccg?catcaccgtg?gtggctgtgc?aggcgtcctc?cgctcagcgg????900

aaggtggagc?gggggcggcg?gatgaagcgc?accaactgcc?tactgatcag?catcgccatc????960

atctttggcg?tttcttggct?gccgctgaac?tttttcaacc?tgtacgcgga?catggagcgc???1020

tcgccggtca?ctcagagcat?gctagtccgc?tacgccatct?gccacatgat?cggcatgagc???1080

tccgcctgct?ccaacccgtt?gctctacggc?tggctcaacg?acaacttccg?taaagaattt???1140

caagaactgc?tctgccgttg?ctcagacact?aatgttgctc?ttaacggtca?cacgacaggc???1200

tgcaacgtcc?aggcggcggc?gcgcaagcgt?cgcaagttgg?gcgccgaact?ctccaaaggc???1260

gaactcaagc?tgctggggcc?aggcggcgcc?cagagcggta?ccgccggcgg?ggaaggcggt???1320

ctggcggcca?ccgacttcat?gaccggccac?cacgagggcg?gactgcgcag?cgccataacc???1380

gagtcggtgg?ccctcacgga?ccacaacccc?gtgccctcgg?aggtcaccaa?gctgatgccg???1440

cggta???????????????????????????????????????????????????????????????1445

<210>4

<211>357

<212>PRT

＜213〉fruit bat (D.melanogaster)

<400>4

Met?Glu?Asn?Thr?Thr?Met?Leu?Ala?Asn?Ile?Ser?Leu?Asn?Ala?Thr?Arg

1???????????????5???????????????????10??????????????????15

Asn?Glu?Glu?Asn?Ile?Thr?Ser?Phe?Phe?Thr?Asp?Glu?Glu?Trp?Leu?Ala

20??????????????????25??????????????????30

Ile?Asn?Gly?Thr?Leu?Pro?Trp?Ile?Val?Gly?Phe?Phe?Phe?Gly?Val?Ile

35??????????????????40??????????????????45

Ala?Ile?Thr?Gly?Phe?Phe?Gly?Asn?Leu?Leu?Val?Ile?Leu?Val?Val?Val

50??????????????????55??????????????????60

Phe?Asn?Asn?Asn?Met?Arg?Ser?Thr?Thr?Asn?Leu?Met?Ile?Val?Asn?Leu

65??????????????????70??????????????????75??????????????????80

Ala?Ala?Ala?Asp?Leu?Met?Phe?Val?Ile?Leu?Cys?Ile?Pro?Phe?Thr?Ala

85??????????????????90??????????????????95

Thr?Asp?Tyr?Met?Val?Tyr?Tyr?Trp?Pro?Tyr?Gly?Arg?Phe?Trp?Cys?Arg

100?????????????????105?????????????????110

Ser?Val?Gln?Tyr?Leu?Ile?Val?Val?Thr?Ala?Phe?Ala?Ser?Ile?Tyr?Thr

115?????????????????120?????????????????125

Leu?Val?Leu?Met?Ser?Ile?Asp?Arg?Phe?Leu?Ala?Val?Val?His?Pro?Ile

130?????????????????135?????????????????140

Arg?Ser?Arg?Met?Met?Arg?Thr?Glu?Asn?Ile?Thr?Leu?Ile?Ala?Ile?Val

145?????????????????150?????????????????155?????????????????160

Thr?Leu?Trp?Ile?Val?Val?Leu?Val?Val?Ser?Val?Pro?Val?Ala?Phe?Thr

165?????????????????170?????????????????175

His?Asp?Val?Val?Val?Asp?Tyr?Asp?Ala?Lys?Lys?Asn?Ile?Thr?Tyr?Gly

180?????????????????185?????????????????190

Met?Cys?Thr?Phe?Thr?Thr?Asn?Asp?Phe?Leu?Gly?Pro?Arg?Thr?Tyr?Gln

195?????????????????200?????????????????205

Val?Thr?Phe?Phe?Ile?Ser?Ser?Tyr?Leu?Leu?Pro?Leu?Met?Ile?Ile?Ser

210?????????????????215?????????????????220

Gly?Leu?Tyr?Met?Arg?Met?Ile?Met?Arg?Leu?Trp?Arg?Gln?Gly?Thr?Gly

225?????????????????230?????????????????235?????????????????240

Val?Arg?Met?Ser?Lys?Glu?Ser?Gln?Arg?Gly?Arg?Lys?Arg?Val?Thr?Arg

245?????????????????250?????????????????255

Leu?Val?Val?Val?Val?Val?Ile?Ala?Phe?Ala?Ser?Leu?Trp?Leu?Pro?Val

260?????????????????265?????????????????270

Gln?Leu?Ile?Leu?Leu?Leu?Lys?Ser?Leu?Asp?Val?Ile?Glu?Thr?Asn?Thr

275?????????????????280?????????????????285

Leu?Thr?Lys?Leu?Val?Ile?Gln?Val?Thr?Ala?Gln?Thr?Leu?Ala?Tyr?Ser

290?????????????????295?????????????????300

Ser?Ser?Cys?Ile?Asn?Pro?Leu?Leu?Tyr?Ala?Phe?Leu?Ser?Glu?Asn?Phe

305?????????????????310?????????????????315?????????????????320

Arg?Lys?Ala?Phe?Tyr?Lys?Ala?Val?Asn?Cys?Ser?Ser?Arg?Tyr?Gln?Asn

325?????????????????330?????????????????335

Tyr?Thr?Ser?Asp?Leu?Pro?Pro?Pro?Arg?Lys?Thr?Ser?Cys?Ala?Arg?Thr

340?????????????????345?????????????????350

Ser?Thr?Thr?Gly?Leu

355

<210>5

<211>1376

<212>DNA

＜213〉fruit bat (D.melanogaster)

<400>5

atgaatcaga?cggagcccgc?ccagctggca?gatggggagc?atctgagtgg?atacgccagc?????60

agcagcaaca?gcgtgcgcta?tctggacgac?cggcatccgc?tggactacct?tgacctgggc????120

acggtgcacg?ccctcaacac?cactgccatc?aacacctcgg?atctgaatga?gactgggagc????180

aggccgctgg?acccggtgct?tatcgatagg?ttcctgagca?acagggcggt?ggacagcccc????240

tggtaccaca?tgctcatcag?catgtacggc?gtgctaatcg?tcttcggcgc?cctaggcaac????300

accctggttg?ttatagccgt?catccggaag?cccatcatgc?gcactgctcg?caatctgttc????360

atcctcaacc?tggccatatc?ggacctactt?ttatgcctag?tcaccatgcc?gctgaccttg????420

atggagatcc?tgtccaagta?ctggccctac?ggctcctgct?ccatcctgtg?caaaacgatt????480

gccatgctgc?aggcactttg?tattttcgtg?tcgacaatat?ccataacggc?cattgccttc????540

gacagatatc?aggtgatcgt?gtaccccacg?cgggacagcc?tgcagttcgt?gggcgcggtg????600

acgatcctgg?cggggatctg?ggcactggca?ctgctgctgg?cctcgccgct?gttcgtctac????660

aaggagctga?tcaacacaga?cacgccggca?ctcctgcagc?agatcggcct?gcaggacacg????720

atcccgtact?gcattgagga?ctggccaagt?cgcaacgggc?gcttctacta?ctcgatcttc????780

tcgctgtgcg?tacaatacct?ggtgcccatc?ctgatcgtct?cggtggcata?cttcgggatc????840

tacaacaagc?tgaagagccg?catcaccgtg?gtggctgtgc?aggcgtcctc?cgctcagcgg????900

aaggtggagc?gggggcggcg?gatgaagcgc?accaactgcc?tactgatcag?catcgccatc????960

atctttggcg?tttcttggct?gccgctgaac?tttttcaacc?tgtacgcgga?catggagcgc???1020

tcgccggtca?ctcagagcat?gctagtccgc?tacgccatct?gccacatgat?cggcatgagc???1080

tccgcctgct?ccaacccgtt?gctctacggc?tggctcaacg?acaacttccg?ctgcaacgtc???1140

caggcggcgg?cgcgcaagcg?tcgcaagttg?ggcgccgaac?tctccaaagg?cgaactcaag???1200

ctgctggggc?caggcggcgc?ccagagcggt?accgccggcg?gggaaggcgg?tctggcggcc???1260

accgacttca?tgaccggcca?ccacgagggc?ggactgcgca?gcgccataac?cgagtcggtg???1320

gccctcacgg?accacaaccc?cgtgccctcg?gaggtcacca?agctgatgcc?gcggta???????1376

<210>6

<211>458

<212>PRT

＜213〉fruit bat (D.melanogaster)

<400>6

Met?Asn?Gln?Thr?Glu?Pro?Ala?Gln?Leu?Ala?Asp?Gly?Glu?His?Leu?Ser

1???????????????5???????????????????10??????????????????15

Gly?Tyr?Ala?Ser?Ser?Ser?Asn?Ser?Val?Arg?Tyr?Leu?Asp?Asp?Arg?His

20??????????????????25??????????????????30

Pro?Leu?Asp?Tyr?Leu?Asp?Leu?Gly?Thr?Val?His?Ala?Leu?Asn?Thr?Thr

35??????????????????40??????????????????45

Ala?Ile?Asn?Thr?Ser?Asp?Leu?Asn?Glu?Thr?Gly?Ser?Arg?Pro?Leu?Asp

50??????????????????55??????????????????60

Pro?Val?Leu?Ile?Asp?Arg?Phe?Leu?Ser?Asn?Arg?Ala?Val?Asp?Ser?Pro

65??????????????????70??????????????????75??????????????????80

Trp?Tyr?His?Met?Leu?Ile?Ser?Met?Tyr?Gly?Val?Leu?Ile?Val?Phe?Gly

85??????????????????90??????????????????95

Ala?Leu?Gly?Asn?Thr?Leu?Val?Val?Ile?Ala?Val?Ile?Arg?Lys?Pro?Ile

100?????????????????105?????????????????110

Met?Arg?Thr?Ala?Arg?Asn?Leu?Phe?Ile?Leu?Asn?Leu?Ala?Ile?Ser?Asp

115?????????????????120?????????????????125

Leu?Leu?Leu?Cys?Leu?Val?Thr?Met?Pro?Leu?Thr?Leu?Met?Glu?Ile?Leu

130?????????????????135?????????????????140

Ser?Lys?Tyr?Trp?Pro?Tyr?Gly?Ser?Cys?Ser?Ile?Leu?Cys?Lys?Thr?Ile

145?????????????????150?????????????????155?????????????????160

Ala?Met?Leu?Gln?Ala?Leu?Cys?Ile?Phe?Val?Ser?Thr?Ile?Ser?Ile?Thr

165?????????????????170?????????????????175

Ala?Ile?Ala?Phe?Asp?Arg?Tyr?Gln?Val?Ile?Val?Tyr?Pro?Thr?Arg?Asp

180?????????????????185?????????????????190

Ser?Leu?Gln?Phe?Val?Gly?Ala?Val?Thr?Ile?Leu?Ala?Gly?Ile?Trp?Ala

195?????????????????200?????????????????205

Leu?Ala?Leu?Leu?Leu?Ala?Ser?Pro?Leu?Phe?Val?Tyr?Lys?Glu?Leu?Ile

210?????????????????215?????????????????220

Asn?Thr?Asp?Thr?Pro?Ala?Leu?Leu?Gln?Gln?Ile?Gly?Leu?Gln?Asp?Thr

225?????????????????230?????????????????235?????????????????240

Ile?Pro?Tyr?Cys?Ile?Glu?Asp?Trp?Pro?Ser?Arg?Asn?Gly?Arg?Phe?Tyr

245?????????????????250?????????????????255

Tyr?Ser?Ile?Phe?Ser?Leu?Cys?Val?Gln?Tyr?Leu?Val?Pro?Ile?Leu?Ile

260?????????????????265?????????????????270

Val?Ser?Val?Ala?Tyr?Phe?Gly?Ile?Tyr?Asn?Lys?Leu?Lys?Ser?Arg?Ile

275?????????????????280?????????????????285

Thr?Val?Val?Ala?Val?Gln?Ala?Ser?Ser?Ala?Gln?Arg?Lys?Val?Glu?Arg

290?????????????????295?????????????????300

Gly?Arg?Arg?Met?Lys?Arg?Thr?Asn?Cys?Leu?Leu?Ile?Ser?Ile?Ala?Ile

305?????????????????310?????????????????315?????????????????320

Ile?Phe?Gly?Val?Ser?Trp?Leu?Pro?Leu?Asn?Phe?Phe?Asn?Leu?Tyr?Ala

325?????????????????330?????????????????335

Asp?Met?Glu?Arg?Ser?Pro?Val?Thr?Gln?Ser?Met?Leu?Val?Arg?Tyr?Ala

340?????????????????345?????????????????350

Ile?Cys?His?Met?Ile?Gly?Met?Ser?Ser?Ala?Cys?Ser?Asn?Pro?Leu?Leu

355?????????????????360?????????????????365

Tyr?Gly?Trp?Leu?Asn?Asp?Asn?Phe?Arg?Cys?Asn?Val?Gln?Ala?Ala?Ala

370?????????????????375?????????????????380

Arg?Lys?Arg?Arg?Lys?Leu?Gly?Ala?Glu?Leu?Ser?Lys?Gly?Glu?Leu?Lys

385?????????????????390?????????????????395?????????????????400

Leu?Leu?Gly?Pro?Gly?Gly?Ala?Gln?Ser?Gly?Thr?Ala?Gly?Gly?Glu?Gly

405?????????????????410?????????????????415

Gly?Leu?Ala?Ala?Thr?Asp?Phe?Met?Thr?Gly?His?His?Glu?Gly?Gly?Leu

420?????????????????425?????????????????430

Arg?Ser?Ala?Ile?Thr?Glu?Ser?Val?Ala?Leu?Thr?Asp?His?Asn?Pro?Val

435?????????????????440?????????????????445

Pro?Ser?Glu?Val?Thr?Lys?Leu?Met?Pro?Arg

450?????????????????455

<210>7

<211>1073

<212>DNA

＜213〉fruit bat (D.melanogaster)

<400>7

atggagaaca?ccacaatgct?ggctaatatt?agcctaaatg?caaccagaaa?tgaggagaat?????60

atcacctcat?tcttcaccga?cgaagagtgg?ctggccatca?atggcacttt?gccgtggata????120

gtgggattct?tcttcggcgt?catcgccatc?acgggattct?tcggcaacct?gctggtcatc????180

ctggtggtgg?tcttcaacaa?caacatgcgc?tccaccacca?acctgatgat?tgtcaatctg????240

gctgccgctg?atctgatgtt?cgtaatcctc?tgcattccct?tcacggccac?cgattacatg????300

gtgtactact?ggccatatgg?aaggttctgg?tgccgcagtg?tccagtacct?gattgtggtg????360

accgccttcg?cctccatcta?cacgctggtg?ctaatgtcca?tcgatcggtt?cctggcggtg????420

gttcatccca?ttcgctcgcg?gatgatgagg?acggagaaca?ttaccctgat?tgccatcgtg????480

actctgtgga?tcgtggtgct?ggtcgtttcg?gtgccagtgg?ccttcaccca?cgacgtggtg????540

gtggactacg?atgcaaagaa?gaacatcacc?tacggcatgt?gcaccttcac?gacgaacgac????600

ttccttggtc?cgcgcaccta?ccaggtcacc?ttcttcatca?gctcctacct?gctgcccctg????660

atgatcatca?gcggtctcta?catgcgcatg?atcatgcggc?tctggcgcca?gggaaccggc????720

gtccgcatgt?ccaaggagtc?gcagcgcggt?cgcaagcggg?tcacccgact?cgtcgtcgtg????780

gtggtcatcg?ccttcgcctc?gctctggctg?cctgtccagc?tcatcctgct?gctcaagtca????840

ctggatgtca?tcgagacgaa?caccctcacc?aagctagtca?tccaggtcac?cgcccagact????900

ctggcctaca?gcagctcgtg?tatcaatccg?ctgctctacg?ccttcctctc?cgagaatttc????960

cggaaggcct?tctataaggc?cgttaactgc?tcctctcgat?accagaacta?cacatctgat???1020

ttgccgccgc?cgcgcaagac?gtcctgtgcc?aggacctcca?ccactggact?cta??????????1073

<210>8

<211>357

<212>PRT

＜213〉fruit bat (D.melanogaster)

<400>8

Met?Glu?Asn?Thr?Thr?Met?Leu?Ala?Asn?Ile?Ser?Leu?Asn?Ala?Thr?Arg

1???????????????5???????????????????10??????????????????15

Asn?Glu?Glu?Asn?Ile?Thr?Ser?Phe?Phe?Thr?Asp?Glu?Glu?Trp?Leu?Ala

20??????????????????25??????????????????30

Ile?Asn?Gly?Thr?Leu?Pro?Trp?Ile?Val?Gly?Phe?Phe?Phe?Gly?Val?Ile

35??????????????????40??????????????????45

Ala?Ile?Thr?Gly?Phe?Phe?Gly?Asn?Leu?Leu?Val?Ile?Leu?Val?Val?Val

50??????????????????55??????????????????60

Phe?Asn?Asn?Asn?Met?Arg?Ser?Thr?Thr?Asn?Leu?Met?Ile?Val?Asn?Leu

65??????????????????70??????????????????75??????????????????80

Ala?Ala?Ala?Asp?Leu?Met?Phe?Val?Ile?Leu?Cys?Ile?Pro?Phe?Thr?Ala

85??????????????????90??????????????????95

Thr?Asp?Tyr?Met?Val?Tyr?Tyr?Trp?Pro?Tyr?Gly?Arg?Phe?Trp?Cys?Arg

100?????????????????105?????????????????110

Ser?Val?Gln?Tyr?Leu?Ile?Val?Val?Thr?Ala?Phe?Ala?Ser?Ile?Tyr?Thr

115?????????????????120?????????????????125

Leu?Val?Leu?Met?Ser?Ile?Asp?Arg?Phe?Leu?Ala?Val?Val?His?Pro?Ile

130????????????????135??????????????????140

Arg?Ser?Arg?Met?Met?Arg?Thr?Glu?Asn?Ile?Thr?Leu?Ile?Ala?Ile?Val

145?????????????????150?????????????????155?????????????????160

Thr?Leu?Trp?Ile?Val?Val?Leu?Val?Val?Ser?Val?Pro?Val?Ala?Phe?Thr

165?????????????????170?????????????????175

His?Asp?Val?Val?Val?Asp?Tyr?Asp?Ala?Lys?Lys?Asn?Ile?Thr?Tyr?Gly

180?????????????????185?????????????????190

Met?Cys?Thr?Phe?Thr?Thr?Asn?Asp?Phe?Leu?Gly?Pro?Arg?Thr?Tyr?Gln

195?????????????????200?????????????????205

Val?Thr?Phe?Phe?Ile?Ser?Ser?Tyr?Leu?Leu?Pro?Leu?Met?Ile?Ile?Ser

210?????????????????215?????????????????220

Gly?Leu?Tyr?Met?Arg?Met?Ile?Met?Arg?Leu?Trp?Arg?Gln?Gly?Thr?Gly

225?????????????????230?????????????????235?????????????????240

Val?Arg?Met?Ser?Lys?Glu?Ser?Gln?Arg?Gly?Arg?Lys?Arg?Val?Thr?Arg

245?????????????????250?????????????????255

Leu?Val?Val?Val?Val?Val?Ile?Ala?Phe?Ala?Ser?Leu?Trp?Leu?Pro?Val

260?????????????????265?????????????????270

Gln?Leu?Ile?Leu?Leu?Leu?Lys?Ser?Leu?Asp?Val?Ile?Glu?Thr?Asn?Thr

275?????????????????280?????????????????285

Leu?Thr?Lys?Leu?Val?Ile?Gln?Val?Thr?Ala?Gln?Thr?Leu?Ala?Tyr?Ser

290?????????????????295?????????????????300

Ser?Ser?Cys?Ile?Asn?Pro?Leu?Leu?Tyr?Ala?Phe?Leu?Ser?Glu?Asn?Phe

305?????????????????310?????????????????315?????????????????320

Arg?Lys?Ala?Phe?Tyr?Lys?Ala?Val?Asn?Cys?Ser?Ser?Arg?Tyr?Gln?Asn

325?????????????????330?????????????????335

Tyr?Thr?Ser?Asp?Leu?Pro?Pro?Pro?Arg?Lys?Thr?Ser?Cys?Ala?Arg?Thr

340?????????????????345?????????????????350

Ser?Thr?Thr?Gly?Leu

355

<210>9

<211>1559

<212>DNA

＜213〉fruit bat (D.melanogaster)

<400>9

atggagaatc?gcagtgactt?cgaggcggat?gactacggcg?acatcagttg?gagcaattgg?????60

agcaactgga?gcacccccgc?cggcgtcctt?ttctcggcca?tgagcagcgt?gctctcggcc????120

agcaaccata?cgcccctgcc?ggactttggc?caggagctcg?ccctatccac?cagctccttc????180

aatcacagcc?agaccctatc?caccgaccag?cccgccgtcg?gggacgtgga?agacgcggcc????240

gaggatgcgg?cggcgtccat?ggagacgggc?tcgtttgcat?ttgtggtccc?gtggtggcgt????300

caggtgctct?ggagcatcct?cttcggcggc?atggtcattg?tggcgacggg?cggtaacctg????360

attgttgtct?ggatcgtgat?gacgaccaag?cggatgcgga?cggtaaccaa?ctatttcata????420

gtgaatctct?ccatcgcgga?cgccatggtg?tccagcctaa?acgtcacctt?caactactac????480

tatatgctgg?atagcgactg?gcccttcggc?gagttctact?gcaagttgtc?ccagttcatc????540

gcgatgctaa?gcatctgcgc?ctcagtgttc?accctaatgg?ccatctccat?cgacagatac????600

gtggccatca?tccggccact?gcagccgcgg?atgagcaagc?ggtgcaacct?ggccatcgcg????660

gcggtcatct?ggctggcctc?cacgctcatc?tcctgcccca?tgatgatcat?ctaccgcacg????720

gaggaggtgc?cggtccgcgg?gctcagcaac?cgcacggtct?gctacccgga?gtggcccgat????780

gggcccacca?atcactccac?gatggagtcc?ctctacaaca?tcctcatcat?catyctaacc????840

tacttcctgc?ccatcgtctc?catgacggtc?acctactcgc?gcgtgggcat?cgagctctgg????900

ggatccaaga?ccatcggcga?gtgcacgccc?cgccaggtgg?araaygtgcg?gagtaagcga????960

agggtggtga?agatgatgat?tgtggtcgtc?ctgatattcg?ccatctgctg?gctgccgttc???1020

cacagctact?tcataatcac?atcctgctac?ccggccatca?cggaggcgcc?cttcatccag???1080

gaactctacc?tggccatcta?ctggctggcc?atgagcaact?ccatgtacaa?tcccattata???1140

tactgctgga?tgaattcgcg?ctttcgctat?ggtttcaaga?tggtcttccg?ctggtgcctg???1200

tttgtgcgcg?tgggcactga?accctttagt?cggcgggaga?acctgacatc?ccggtactcc???1260

tgctccggtt?ccccggatca?caatcgcatc?aagcgcaatg?atacccagaa?atcgatactt???1320

tatacctgtc?ccagctcacc?caagtcgcat?cgaatttcgc?acagcggaac?aggtcgcagt???1380

gcgacgctgc?ggaacagtct?gccggcggag?tcactgtcgt?ccggcggatc?tggtggtgga???1440

gggcacagga?aacggttgtc?ctaccagcag?gaaatgcagc?agcgttggtc?aggacccaat???1500

agtgccaccg?cagtgaccaa?ttccagcagt?acggccaaca?ccacccaact?gctctcctg????1559

<210>10

<211>519

<212>PRT

＜213〉fruit bat (D.melanogaster)

<400>10

Met?Glu?Asn?Arg?Ser?Asp?Phe?Glu?Ala?Asp?Asp?Tyr?Gly?Asp?Ile?Ser

1???????????????5???????????????????10??????????????????15

Trp?Ser?Asn?Trp?Ser?Asn?Trp?Ser?Thr?Pro?Ala?Gly?Val?Leu?Phe?Ser

20??????????????????25??????????????????30

Ala?Met?Ser?Ser?Val?Leu?Ser?Ala?Ser?Asn?His?Thr?Pro?Leu?Pro?Asp

35??????????????????40??????????????????45

Phe?Gly?Gln?Glu?Leu?Ala?Leu?Ser?Thr?Ser?Ser?Phe?Asn?His?Ser?Gln

50??????????????????55??????????????????60

Thr?Leu?Ser?Thr?Asp?Gln?Pro?Ala?Val?Gly?Asp?Val?Glu?Asp?Ala?Ala

65??????????????????70??????????????????75??????????????????80

Glu?Asp?Ala?Ala?Ala?Ser?Met?Glu?Thr?Gly?Ser?Phe?Ala?Phe?Val?Val

85??????????????????90??????????????????95

Pro?Trp?Trp?Arg?Gln?Val?Leu?Trp?Ser?Ile?Leu?Phe?Gly?Gly?Met?Val

100?????????????????105?????????????????110

Ile?Val?Ala?Thr?Gly?Gly?Asn?Leu?Ile?Val?Val?Trp?Ile?Val?Met?Thr

115?????????????????120?????????????????125

Thr?Lys?Arg?Met?Arg?Thr?Val?Thr?Asn?Tyr?Phe?Ile?Val?Asn?Leu?Ser

130?????????????????135?????????????????140

Ile?Ala?Asp?Ala?Met?Val?Ser?Ser?Leu?Asn?Val?Thr?Phe?Asn?Tyr?Tyr

145?????????????????150?????????????????155?????????????????160

Tyr?Met?Leu?Asp?Ser?Asp?Trp?Pro?Phe?Gly?Glu?Phe?Tyr?Cys?Lys?Leu

165?????????????????170?????????????????175

Ser?Gln?Phe?Ile?Ala?Met?Leu?Ser?Ile?Cys?Ala?Ser?Val?Phe?Thr?Leu

180?????????????????185?????????????????190

Met?Ala?Ile?Ser?Ile?Asp?Arg?Tyr?Val?Ala?Ile?Ile?Arg?Pro?Leu?Gln

195?????????????????200?????????????????205

Pro?Arg?Met?Ser?Lys?Arg?Cys?Asn?Leu?Ala?Ile?Ala?Ala?Val?Ile?Trp

210?????????????????215?????????????????220

Leu?Ala?Ser?Thr?Leu?Ile?Ser?Cys?Pro?Met?Met?Ile?Ile?Tyr?Arg?Thr

225?????????????????230?????????????????235?????????????????240

Glu?Glu?Val?Pro?Val?Arg?Gly?Leu?Ser?Asn?Arg?Thr?Val?Cys?Tyr?Pro

245?????????????????250?????????????????255

Glu?Trp?Pro?Asp?Gly?Pro?Thr?Asn?His?Ser?Thr?Met?Glu?Ser?Leu?Tyr

260?????????????????265?????????????????270

Asn?Ile?Leu?Ile?Ile?Ile?Leu?Thr?Tyr?Phe?Leu?Pro?Ile?Val?Ser?Met

275?????????????????280?????????????????285

Thr?Val?Thr?Tyr?Ser?Arg?Val?Gly?Ile?Glu?Leu?Trp?Gly?Ser?Lys?Thr

290?????????????????295?????????????????300

Ile?Gly?Glu?Cys?Thr?Pro?Arg?Gln?Val?Glu?Asn?Val?Arg?Ser?Lys?Arg

305?????????????????310?????????????????315?????????????????320

Arg?Val?Val?Lys?Met?Met?Ile?Val?Val?Val?Leu?Ile?Phe?Ala?Ile?Cys

325?????????????????330?????????????????335

Trp?Leu?Pro?Phe?His?Ser?Tyr?Phe?Ile?Ile?Thr?Ser?Cys?Tyr?Pro?Ala

340?????????????????345?????????????????350

Ile?Thr?Glu?Ala?Pro?Phe?Ile?Gln?Glu?Leu?Tyr?Leu?Ala?Ile?Tyr?Trp

355?????????????????360?????????????????365

Leu?Ala?Met?Ser?Asn?Ser?Met?Tyr?Asn?Pro?Ile?Ile?Tyr?Cys?Trp?Met

370?????????????????375?????????????????380

Asn?Ser?Arg?Phe?Arg?Tyr?Gly?Phe?Lys?Met?Val?Phe?Arg?Trp?Cys?Leu

385?????????????????390?????????????????395?????????????????400

Phe?Val?Arg?Val?Gly?Thr?Glu?Pro?Phe?Ser?Arg?Arg?Glu?Asn?Leu?Thr

405?????????????????410?????????????????415

Ser?Arg?Tyr?Ser?Cys?Ser?Gly?Ser?Pro?Asp?His?Asn?Arg?Ile?Lys?Arg

420?????????????????425?????????????????430

Asn?Asp?Thr?Gln?Lys?Ser?Ile?Leu?Tyr?Thr?Cys?Pro?Ser?Ser?Pro?Lys

435?????????????????440?????????????????445

Ser?His?Arg?Ile?Ser?His?Ser?Gly?Thr?Gly?Arg?Ser?Ala?Thr?Leu?Arg

450?????????????????455?????????????????460

Asn?Ser?Leu?Pro?Ala?Glu?Ser?Leu?Ser?Ser?Gly?Gly?Ser?Gly?Gly?Gly

465?????????????????470?????????????????475?????????????????480

Gly?His?Arg?Lys?Arg?Leu?Ser?Tyr?Gln?Gln?Glu?Met?Gln?Gln?Arg?Trp

485?????????????????490?????????????????495

Ser?Gly?Pro?Asn?Ser?Ala?Thr?Ala?Val?Thr?Asn?Ser?Ser?Ser?Thr?Ala

500?????????????????505?????????????????510

Asn?Thr?Thr?Gln?Leu?Leu?Ser

515

<210>11

<211>1568

<212>DNA

＜213〉fruit bat (D.melanogaster)

<400>11

atggagaatc?gcagtgactt?cgaggcggat?gactacggcg?acatcagttg?gagcaattgg?????60

agcaattgga?gcaactggag?cacccccgcc?ggcgtccttt?tctcggccat?gagcagcgtg????120

ctctcggcca?gcaaccatac?gcctctgccg?gactttggcc?aggagctcgc?cctatccacc????180

agctccttca?atcacagcca?gaccctatcc?accgacctgc?ccgccgtcgg?ggacgtggaa????240

gacgcggccg?aggatgcggc?ggcgtccatg?gagacgggct?cgtttgcatt?tgtggtcccg????300

tggtggcgtc?aggtgctctg?gagcatcctc?ttcggcggca?tggtcattgt?ggcgacgggc????360

ggtaacctga?ttgttgtctg?gatcgtgatg?acgaccaagc?ggatgcggac?ggtaaccaac????420

tatttcatag?taaatctctc?catcgcggac?gccatggtgt?ccagcctgaa?cgtcaccttc????480

aactactact?acatgctgga?tagcgactgg?cccttcggcg?agttctactg?caagttgtcc????540

cagttcatcg?cgatgctaag?catctgcgcc?tcagtgttca?ccctaatggc?catctccatc????600

gacagatacg?tggccatcat?ccggccactg?cagccgcgga?tgagcaagcg?gtgcaacctg????660

gccatcgcgg?cggtcatctg?gctggcctcc?acgctcatct?cctgccccat?gatgatcatc????720

taccgcacgg?aggaggtgcc?ggtccgcggg?ctcagcaacc?gcacggtctg?ctacccggag????780

tggcccgatg?ggcccaccaa?tcactccacg?atggagtccc?tctacaacat?cctcatcatc????840

attctaacct?acttcctgcc?catcgtctcc?atgacggtca?cctactcgcg?cgtgggcatc????900

gagctctggg?gatccaagac?catcggcgag?tgcacgcccc?gccaggtgga?gaatgtgcgg????960

agtaagcgaa?gggtggtgaa?gatgatgatt?gtggtcgtcc?tgatattcgc?catctgctgg???1020

ctgccgttcc?acagctactt?cataatcaca?tcctgctacc?cggccatcac?ggaggcgccc???1080

ttcatccagg?aactttacct?ggccatctac?tggctggcca?tgagcaactc?catgtacaat???1140

cccattatat?actgctggat?gaattcgcgc?tttcgctatg?gtttcaagat?ggtcttccgc???1200

tggtgcctgt?ttgtgcgcgt?gggcactgaa?ccctttagtc?ggcgggagaa?cctgacatcc???1260

cggtactcct?gctccggttc?cccggatcac?aatcgcatca?agcgcaatga?tacccagaaa???1320

tcgatacttt?atacctgtcc?cagctcaccc?aagtcgcatc?gaatttcgca?cagcggaaca???1380

ggtcgcagtg?cgacgctgag?gaacagtctg?ccggcggagt?cattgtcgtc?cggtggatct???1440

ggaggtggag?gacacaggaa?acggttgtcc?taccagcagg?aaatgcagca?gcggtggtca???1500

ggacccaata?gtgccaccgc?agtgaccaat?tccagcagta?cggccaacac?cacccaactg???1560

ctctcctg????????????????????????????????????????????????????????????1568

<210>12

<211>522

<212>PRT

＜213〉fruit bat (D.melanogaster)

<400>12

Met?Glu?Asn?Arg?Ser?Asp?Phe?Glu?Ala?Asp?Asp?Tyr?Gly?Asp?Ile?Ser

1???????????????5???????????????????10??????????????????15

Trp?Ser?Asn?Trp?Ser?Asn?Trp?Ser?Asn?Trp?Ser?Thr?Pro?Ala?Gly?Val

20??????????????????25??????????????????30

Leu?Phe?Ser?Ala?Met?Ser?Ser?Val?Leu?Ser?Ala?Ser?Asn?His?Thr?Pro

35??????????????????40??????????????????45

Leu?Pro?Asp?Phe?Gly?Gln?Glu?Leu?Ala?Leu?Ser?Thr?Ser?Ser?Phe?Asn

50??????????????????55??????????????????60

His?Ser?Gln?Thr?Leu?Ser?Thr?Asp?Leu?Pro?Ala?Val?Gly?Asp?Val?Glu

65??????????????????70??????????????????75??????????????????80

Asp?Ala?Ala?Glu?Asp?Ala?Ala?Ala?Ser?Met?Glu?Thr?Gly?Ser?Phe?Ala

85??????????????????90??????????????????95

Phe?Val?Val?Pro?Trp?Trp?Arg?Gln?Val?Leu?Trp?Ser?Ile?Leu?Phe?Gly

100?????????????????105?????????????????110

Gly?Met?Val?Ile?Val?Ala?Thr?Gly?Gly?Asn?Leu?Ile?Val?Val?Trp?Ile

115?????????????????120?????????????????125

Val?Met?Thr?Thr?Lys?Arg?Met?Arg?Thr?Val?Thr?Asn?Tyr?Phe?Ile?Val

130?????????????????135?????????????????140

Asn?Leu?Ser?Ile?Ala?Asp?Ala?Met?Val?Ser?Ser?Leu?Asn?Val?Thr?Phe

145?????????????????150?????????????????155?????????????????160

Asn?Tyr?Tyr?Tyr?Met?Leu?Asp?Ser?Asp?Trp?Pro?Phe?Gly?Glu?Phe?Tyr

165?????????????????170?????????????????175

Cys?Lys?Leu?Ser?Gln?Phe?Ile?Ala?Met?Leu?Ser?Ile?Cys?Ala?Ser?Val

180?????????????????185?????????????????190

Phe?Thr?Leu?Met?Ala?Ile?Ser?Ile?Asp?Arg?Tyr?Val?Ala?Ile?Ile?Arg

195?????????????????200?????????????????205

Pro?Leu?Gln?Pro?Arg?Met?Ser?Lys?Arg?Cys?Asn?Leu?Ala?Ile?Ala?Ala

210?????????????????215?????????????????220

Val?Ile?Trp?Leu?Ala?Ser?Thr?Leu?Ile?Ser?Cys?Pro?Met?Met?Ile?Ile

225?????????????????230?????????????????235?????????????????240

Tyr?Arg?Thr?Glu?Glu?Val?Pro?Val?Arg?Gly?Leu?Ser?Asn?Arg?Thr?Val

245?????????????????250?????????????????255

Cys?Tyr?Pro?Glu?Trp?Pro?Asp?Gly?Pro?Thr?Asn?His?Ser?Thr?Met?Glu

260?????????????????265?????????????????270

Ser?Leu?Tyr?Asn?Ile?Leu?Ile?Ile?Ile?Leu?Thr?Tyr?Phe?Leu?Pro?Ile

275?????????????????280?????????????????285

Val?Ser?Met?Thr?Val?Thr?Tyr?Ser?Arg?Val?Gly?Ile?Glu?Leu?Trp?Gly

290?????????????????295?????????????????300

Ser?Lys?Thr?Ile?Gly?Glu?Cys?Thr?Pro?Arg?Gln?Val?Glu?Asn?Val?Arg

305?????????????????310?????????????????315?????????????????320

Ser?Lys?Arg?Arg?Val?Val?Lys?Met?Met?Ile?Val?Val?Val?Leu?Ile?Phe

325?????????????????330?????????????????335

Ala?Ile?Cys?Trp?Leu?Pro?Phe?His?Ser?Tyr?Phe?Ile?Ile?Thr?Ser?Cys

340?????????????????345?????????????????350

Tyr?Pro?Ala?Ile?Thr?Glu?Ala?Pro?Phe?Ile?Gln?Glu?Leu?Tyr?Leu?Ala

355?????????????????360?????????????????365

Ile?Tyr?Trp?Leu?Ala?Met?Ser?Asn?Ser?Met?Tyr?Asn?Pro?Ile?Ile?Tyr

370?????????????????375?????????????????380

Cys?Trp?Met?Asn?Ser?Arg?Phe?Arg?Tyr?Gly?Phe?Lys?Met?Val?Phe?Arg

385?????????????????390?????????????????395?????????????????400

Trp?Cys?Leu?Phe?Val?Arg?Val?Gly?Thr?Glu?Pro?Phe?Ser?Arg?Arg?Glu

405?????????????????410?????????????????415

Asn?Leu?Thr?Ser?Arg?Tyr?Ser?Cys?Ser?Gly?Ser?Pro?Asp?His?Asn?Arg

420?????????????????425?????????????????430

Ile?Lys?Arg?Asn?Asp?Thr?Gln?Lys?Ser?Ile?Leu?Tyr?Thr?Cys?Pro?Ser

435?????????????????440?????????????????445

Ser?Pro?Lys?Ser?His?Arg?Ile?Ser?His?Ser?Gly?Thr?Gly?Arg?Ser?Ala

450?????????????????455?????????????????460

Thr?Leu?Arg?Asn?Ser?Leu?Pro?Ala?Glu?Ser?Leu?Ser?Ser?Gly?Gly?Ser

465?????????????????470?????????????????475?????????????????480

Gly?Gly?Gly?Gly?His?Arg?Lys?Arg?Leu?Ser?Tyr?Gln?Gln?Glu?Met?Gln

485?????????????????490?????????????????495

Gln?Arg?Trp?Ser?Gly?Pro?Asn?Ser?Ala?Thr?Ala?Val?Thr?Asn?Ser?Ser

500?????????????????505?????????????????510

Ser?Thr?Ala?Asn?Thr?Thr?Gln?Leu?Leu?Ser

515?????????????????520

<210>13

<211>1394

<212>DNA

＜213〉fruit bat (D.melanogaster)

<400>13

atggagcacc?acaatagcca?tctgttgcct?ggtggcagcg?agaagatgta?ctacatagct?????60

caccagcagc?cgatgctgcg?gaacgaggat?gataactacc?aggaggggta?cttcatcagg????120

ccggaccctg?catccttact?ttacaatacc?accgcactgc?cagcggacga?tgaagggtcc????180

aactatggat?atggctccac?cacaacgctc?agtggcctcc?agttcgagac?ctataatatc????240

actgtgatga?tgaactttag?ctgtgacgac?tatgaccttc?tatcggagga?catgtggtct????300

agtgcctact?ttaagatcat?cgtctacatg?ctctacattc?ccatctttat?cttcgccctg????360

atcggcaacg?gaacggtctg?ctatatcgtc?tattccacac?ctcgcatgcg?cacggtcacc????420

aattacttta?tagccagctt?ggccatcggc?gacatcctga?tgtccttctt?ctgcgttccg????480

tcgtccttca?tctcgctgtt?catcctgaac?tactggcctt?ttggcctggc?cctctgtcac????540

tttgtgaact?actcgcaggc?ggtctcagtt?ctggtcagcg?cctatacttt?ggtggcaatt????600

agcattgacc?gctacatagc?cattatgtgg?ccattaaagc?cacgcatcac?aaaacgctat????660

gccaccttca?tcatcgccgg?cgtttggttt?attgcacttg?ccaccgcact?tcccataccc????720

atcgtctctg?gactcgacat?cccaatgtcg?ccgtggcaca?cgaaatgcga?gaaatacatt????780

tgccgcgaaa?tgtggccgtc?gcggacgcag?gagtactact?acaccctgtc?cctcttcgcg????840

ctgcagttcg?tcgtgccgct?gggcgtgctc?atcttcacct?acgcccggat?caccattcgc????900

gtctgggcga?aacgaccgcc?aggcgaggcg?gaaaccaacc?gcgaccagcg?gatggcacgc????960

tccaaacgga?agatggtcaa?aatgatgctg?acggttgtga?ttgtgttcac?ctgctgttgg???1020

ctgcccttca?atattttgca?gcttttactg?aacgacgagg?agttcgccca?ctgggatcct???1080

ctgccgtatg?tatggttcgc?gtttcactgg?ctggccatgt?cgcactgctg?ctacaatccg???1140

atcatctact?gctacatgaa?cgcccgtttc?aggagcggat?tcgtccagct?gatgcaccgt???1200

atgcccggcc?tgcgtcgctg?gtgctgcctg?cggagcgtcg?gtgatcgcat?gaacgcaact???1260

tccggaacgg?gtccagcact?tcctctcaat?cgaatgaaca?catccaccac?ctacatcagc???1320

gctcgtcgaa?agccacgagc?gacatctttg?cgagcgaacc?cattatcatg?cggcgagacg???1380

tcaccactgc?ggta?????????????????????????????????????????????????????1394

<210>14

<211>464

<212>PRT

＜213〉fruit bat (D.melanogaster)

<400>14

Met?Glu?His?His?Asn?Ser?His?Leu?Leu?Pro?Gly?Gly?Ser?Glu?Lys?Met

1???????????????5???????????????????10??????????????????15

Tyr?Tyr?Ile?Ala?His?Gln?Gln?Pro?Met?Leu?Arg?Asn?Glu?Asp?Asp?Asn

20??????????????????25??????????????????30

Tyr?Gln?Glu?Gly?Tyr?Phe?Ile?Arg?Pro?Asp?Pro?Ala?Ser?Leu?Leu?Tyr

35??????????????????40??????????????????45

Asn?Thr?Thr?Ala?Leu?Pro?Ala?Asp?Asp?Glu?Gly?Ser?Asn?Tyr?Gly?Tyr

50??????????????????55??????????????????60

Gly?Ser?Thr?Thr?Thr?Leu?Ser?Gly?Leu?Gln?Phe?Glu?Thr?Tyr?Asn?Ile

65??????????????????70??????????????????75??????????????????80

Thr?Val?Met?Met?Asn?Phe?Ser?Cys?Asp?Asp?Tyr?Asp?Leu?Leu?Ser?Glu

85??????????????????90??????????????????95

Asp?Met?Trp?Ser?Ser?Ala?Tyr?Phe?Lys?Ile?Ile?Val?Tyr?Met?Leu?Tyr

100?????????????????105?????????????????110

Ile?Pro?Ile?Phe?Ile?Phe?Ala?Leu?Ile?Gly?Asn?Gly?Thr?Val?Cys?Tyr

115?????????????????120?????????????????125

Ile?Val?Tyr?Ser?Thr?Pro?Arg?Met?Arg?Thr?Val?Thr?Asn?Tyr?Phe?Ile

130?????????????????135?????????????????140

Ala?Ser?Leu?Ala?Ile?Gly?Asp?Ile?Leu?Met?Ser?Phe?Phe?Cys?Val?Pro

145?????????????????150?????????????????155?????????????????160

Ser?Ser?Phe?Ile?Ser?Leu?Phe?Ile?Leu?Asn?Tyr?Trp?Pro?Phe?Gly?Leu

165?????????????????170?????????????????175

Ala?Leu?Cys?His?Phe?Val?Asn?Tyr?Ser?Gln?Ala?Val?Ser?Val?Leu?Val

180?????????????????185?????????????????190

Ser?Ala?Tyr?Thr?Leu?Val?Ala?Ile?Ser?Ile?Asp?Arg?Tyr?Ile?Ala?Ile

195?????????????????200?????????????????205

Met?Trp?Pro?Leu?Lys?Pro?Arg?Ile?Thr?Lys?Arg?Tyr?Ala?Thr?Phe?Ile

210?????????????????215?????????????????220

Ile?Ala?Gly?Val?Trp?Phe?Ile?Ala?Leu?Ala?Thr?Ala?Leu?Pro?Ile?Pro

225?????????????????230?????????????????235?????????????????240

Ile?Val?Ser?Gly?Leu?Asp?Ile?Pro?Met?Ser?Pro?Trp?His?Thr?Lys?Cys

245?????????????????250?????????????????255

Glu?Lys?Tyr?Ile?Cys?Arg?Glu?Met?Trp?Pro?Ser?Arg?Thr?Gln?Glu?Tyr

260?????????????????265?????????????????270

Tyr?Tyr?Thr?Leu?Ser?Leu?Phe?Ala?Leu?Gln?Phe?Val?Val?Pro?Leu?Gly

275?????????????????280?????????????????285

Val?Leu?Ile?Phe?Thr?Tyr?Ala?Arg?Ile?Thr?Ile?Arg?Val?Trp?Ala?Lys

290?????????????????295?????????????????300

Arg?Pro?Pro?Gly?Glu?Ala?Glu?Thr?Asn?Arg?Asp?Gln?Arg?Met?Ala?Arg

305?????????????????310?????????????????315?????????????????320

Ser?Lys?Arg?Lys?Met?Val?Lys?Met?Met?Leu?Thr?Val?Val?Ile?Val?Phe

325?????????????????330?????????????????335

Thr?Cys?Cys?Trp?Leu?Pro?Phe?Asn?Ile?Leu?Gln?Leu?Leu?Leu?Asn?Asp

340?????????????????345?????????????????350

Glu?Glu?Phe?Ala?His?Trp?Asp?Pro?Leu?Pro?Tyr?Val?Trp?Phe?Ala?Phe

355?????????????????360?????????????????365

His?Trp?Leu?Ala?Met?Ser?His?Cys?Cys?Tyr?Asn?Pro?Ile?Ile?Tyr?Cys

370?????????????????375?????????????????380

Tyr?Met?Asn?Ala?Arg?Phe?Arg?Ser?Gly?Phe?Val?Gln?Leu?Met?His?Arg

385?????????????????390?????????????????395?????????????????400

Met?Pro?Gly?Leu?Arg?Arg?Trp?Cys?Cys?Leu?Arg?Ser?Val?Gly?Asp?Arg

405?????????????????410?????????????????415

Met?Asn?Ala?Thr?Ser?Gly?Thr?Gly?Pro?Ala?Leu?Pro?Leu?Asn?Arg?Met

420?????????????????425?????????????????430

Asn?Thr?Ser?Thr?Thr?Tyr?Ile?Ser?Ala?Arg?Arg?Lys?Pro?Arg?Ala?Thr

435?????????????????440?????????????????445

Ser?Leu?Arg?Ala?Asn?Pro?Leu?Ser?Cys?Gly?Glu?Thr?Ser?Pro?Leu?Arg

450?????????????????455?????????????????460

<210>15

<211>1556

<212>DNA

＜213〉fruit bat (D.melanogaster)

<400>15

atggagcacc?acaatagcca?tctgttgcct?ggtggcagcg?agaagatgta?ctacatagct?????60

caccagcagc?cgatgctgcg?gaacgaggat?gataactacc?aggaggggta?cttcatcagg????120

ccggaccctg?catccttact?ttacaatacc?accgcactgc?cagcggacga?tgaagggtcc????180

aactatggat?atggctccac?cacaacgctc?agtggcctcc?agttcgagac?ctataatatc????240

actgtgatga?tgaactttag?ctgtgacgac?tatgaccttc?tatcggagga?catgtggtct????300

agtgcctact?ttaagatcat?cgtctacatg?ctctacattc?ccatctttat?cttcgccctg????360

atcggcaacg?gaacggtctg?ctatatcgtc?tattccacac?ctcgcatgcg?cacggtcacc????420

aattacttta?tagccagctt?ggccatcggc?gacatcctga?tgtccttctt?ctgcgttccg????480

tcgtccttca?tctcgctgtt?catcctgaac?tactggcctt?ttggcctggc?cctctgtcac????540

tttgtgaact?actcgcaggc?ggtctcagtt?ctggtcagcg?cctatacttt?ggtggcaatt????600

agcattgacc?gctacatagc?cattatgtgg?ccattaaagc?cacgcatcac?aaaacgctat????660

gccaccttca?tcatcgccgg?cgtttggttt?attgcacttg?ccaccgcact?tcccataccc????720

atcgtctctg?gactcgacat?cccaatgtcg?ccgtggcaca?cgaaatgcga?gaaatacatt????780

tgccgcgaaa?tgtggccgtc?gcggacgcag?gagtactact?acaccctgtc?cctcttcgcg????840

ctgcagttcg?tcgtgccgct?gggcgtgctc?atcttcacct?acgcccggat?caccattcgc????900

gtctgggcga?aacgaccgcc?aggcgaggcg?gaaaccaacc?gcgaccagcg?gatggcacgc????960

tccaaacgga?agatggtcaa?aatgatgctg?acggttgtga?ttgtgttcac?ctgctgttgg???1020

ctgcccttca?atattttgca?gcttttactg?aacgacgagg?agttcgccca?ctgggatcct???1080

ctgccgtatg?tgtggttcgc?gtttcactgg?ctggccatgt?cgcactgctg?ctacaatccg???1140

atcatctact?gctacatgaa?cgcccgtttc?aggagcggat?tcgtccagct?gatgcaccgt???1200

atgcccggcc?tgcgtcgctg?gtgctgcctg?cggagcgtcg?gtgatcgcat?gaacgcaact???1260

tccggtgaga?tgactacgaa?gtaccatcgc?catgtcggcg?atgccctatt?ccggaaaccc???1320

aaaatatgca?ttaggaacgg?gtccagcact?tcctctcaat?cgaatgaaca?catccaccac???1380

ctacatcagc?gctcgtcgaa?agccacgagc?gacatctttg?cgagcgaacc?cattatcatg???1440

cggcgagacg?tcaccactgc?ggtagctgtc?atatcaaaaa?ataaaactga?ttcaccggtg???1500

cgccgatcgg?gaagctcagg?tggaacagaa?gcaaacataa?gaagcaccga?gttttg???????1556

<210>16

<211>518

<212>PRT

＜213〉fruit bat (D.melanogaster)

<400>16

Met?Glu?His?His?Asn?Ser?His?Leu?Leu?Pro?Gly?Gly?Ser?Glu?Lys?Met

1???????????????5???????????????????10??????????????????15

Tyr?Tyr?Ile?Ala?His?Gln?Gln?Pro?Met?Leu?Arg?Asn?Glu?Asp?Asp?Asn

20??????????????????25??????????????????30

Tyr?Gln?Glu?Gly?Tyr?Phe?Ile?Arg?Pro?Asp?Pro?Ala?Ser?Leu?Leu?Tyr

35??????????????????40??????????????????45

Asn?Thr?Thr?Ala?Leu?Pro?Ala?Asp?Asp?Glu?Gly?Ser?Asn?Tyr?Gly?Tyr

50??????????????????55??????????????????60

Gly?Ser?Thr?Thr?Thr?Leu?Ser?Gly?Leu?Gln?Phe?Glu?Thr?Tyr?Asn?Ile

65??????????????????70??????????????????75??????????????????80

Thr?Val?Met?Met?Asn?Phe?Ser?Cys?Asp?Asp?Tyr?Asp?Leu?Leu?Ser?Glu

85??????????????????90??????????????????95

Asp?Met?Trp?Ser?Ser?Ala?Tyr?Phe?Lys?Ile?Ile?Val?Tyr?Met?Leu?Tyr

100?????????????????105?????????????????110

Ile?Pro?Ile?Phe?Ile?Phe?Ala?Leu?Ile?Gly?Asn?Gly?Thr?Val?Cys?Tyr

115?????????????????120?????????????????125

Ile?Val?Tyr?Ser?Thr?Pro?Arg?Met?Arg?Thr?Val?Thr?Asn?Tyr?Phe?Ile

130?????????????????135?????????????????140

Ala?Ser?Leu?Ala?Ile?Gly?Asp?Ile?Leu?Met?Ser?Phe?Phe?Cys?Val?Pro

145?????????????????150?????????????????155?????????????????160

Ser?Ser?Phe?Ile?Ser?Leu?Phe?Ile?Leu?Asn?Tyr?Trp?Pro?Phe?Gly?Leu

165?????????????????170?????????????????175

Ala?Leu?Cys?His?Phe?Val?Asn?Tyr?Ser?Gln?Ala?Val?Ser?Val?Leu?Val

180?????????????????185?????????????????190

Ser?Ala?Tyr?Thr?Leu?Val?Ala?Ile?Ser?Ile?Asp?Arg?Tyr?Ile?Ala?Ile

195?????????????????200?????????????????205

Met?Trp?Pro?Leu?Lys?Pro?Arg?Ile?Thr?Lys?Arg?Tyr?Ala?Thr?Phe?Ile

210?????????????????215?????????????????220

Ile?Ala?Gly?Val?Trp?Phe?Ile?Ala?Leu?Ala?Thr?Ala?Leu?Pro?Ile?Pro

225?????????????????230?????????????????235?????????????????240

Ile?Val?Ser?Gly?Leu?Asp?Ile?Pro?Met?Ser?Pro?Trp?His?Thr?Lys?Cys

245?????????????????250?????????????????255

Glu?Lys?Tyr?Ile?Cys?Arg?Glu?Met?Trp?Pro?Ser?Arg?Thr?Gln?Glu?Tyr

260?????????????????265?????????????????270

Tyr?Tyr?Thr?Leu?Ser?Leu?Phe?Ala?Leu?Gln?Phe?Val?Val?Pro?Leu?Gly

275?????????????????280?????????????????285

Val?Leu?Ile?Phe?Thr?Tyr?Ala?Arg?Ile?Thr?Ile?Arg?Val?Trp?Ala?Lys

290?????????????????295?????????????????300

Arg?Pro?Pro?Gly?Glu?Ala?Glu?Thr?Asn?Arg?Asp?Gln?Arg?Met?Ala?Arg

305?????????????????310?????????????????315?????????????????320

Ser?Lys?Arg?Lys?Met?Val?Lys?Met?Met?Leu?Thr?Val?Val?Ile?Val?Phe

325?????????????????330?????????????????335

Thr?Cys?Cys?Trp?Leu?Pro?Phe?Asn?Ile?Leu?Gln?Leu?Leu?Leu?Asn?Asp

340?????????????????345?????????????????350

Glu?Glu?Phe?Ala?His?Trp?Asp?Pro?Leu?Pro?Tyr?Val?Trp?Phe?Ala?Phe

355?????????????????360?????????????????365

His?Trp?Leu?Ala?Met?Ser?His?Cys?Cys?Tyr?Asn?Pro?Ile?Ile?Tyr?Cys

370?????????????????375?????????????????380

Tyr?Met?Asn?Ala?Arg?Phe?Arg?Ser?Gly?Phe?Val?Gln?Leu?Met?His?Arg

385?????????????????390?????????????????395?????????????????400

Met?Pro?Gly?Leu?Arg?Arg?Trp?Cys?Cys?Leu?Arg?Ser?Val?Gly?Asp?Arg

405?????????????????410?????????????????415

Met?Asn?Ala?Thr?Ser?Gly?Glu?Met?Thr?Thr?Lys?Tyr?His?Arg?His?Val

420?????????????????425?????????????????430

Gly?Asp?Ala?Leu?Phe?Arg?Lys?Pro?Lys?Ile?Cys?Ile?Arg?Asn?Gly?Ser

435?????????????????440?????????????????445

Ser?Thr?Ser?Ser?Gln?Ser?Asn?Glu?His?Ile?His?His?Leu?His?Gln?Arg

450?????????????????455?????????????????460

Ser?Ser?Lys?Ala?Thr?Ser?Asp?Ile?Phe?Ala?Ser?Glu?Pro?Ile?Ile?Met

465?????????????????470?????????????????475?????????????????480

Arg?Arg?Asp?Val?Thr?Thr?Ala?Val?Ala?Val?Ile?Ser?Lys?Asn?Lys?Thr

485?????????????????490?????????????????495

Asp?Ser?Pro?Val?Arg?Arg?Ser?Gly?Ser?Ser?Gly?Gly?Thr?Glu?Ala?Asn

500?????????????????505?????????????????510

Ile?Arg?Ser?Thr?Glu?Phe

515

<210>17

<211>1628

<212>DNA

＜213〉fruit bat (D.melanogaster)

<400>17

atggcaatgg?acttaatcga?gcaggagtcc?cgcctggaat?tcctgcccgg?agccgaggag?????60

gaagcagaat?ttgagcgtct?atacgcggct?cccgctgaga?ttgtggccct?gttgtccatt????120

ttctatgggg?gaatcagtat?cgtggccgtc?attggcaaca?ctttggtcat?ctgggtggtg????180

gccacgacca?ggcaaatgcg?gaccgtgaca?aatatgtata?tcgctaattt?ggcttttgcc????240

gatgtgatta?ttggcctctt?ctgcatacca?tttcagttcc?aggctgccct?gctgcagagt????300

tggaacctgc?cgtggttcat?gtgcagcttc?tgccccttcg?tccaggccct?gagtgtaaat????360

gtctcggtat?tcacgctgac?cgccattgca?atcgatcggc?atagggccat?cattaatcca????420

cttagggcac?gtcccaccaa?gttcgtatcg?aagttcataa?ttggtggaat?ttggatgctg????480

gccctgctat?ttgcggtgcc?ctttgccatt?gcctttcgtg?tggaggagtt?gaccgaaaga????540

tttcgcgaga?acaatgagac?ctacaatgtg?acgcggccat?tctgcatgaa?caagaaccta????600

tccgatgatc?aattgcaatc?ctttcgctac?accctggttt?ttgtgcagta?tctggttcca????660

ttctgtgtca?tcagctttgt?ctacatccag?atggcggtac?gattgtgggg?cacacgtgct????720

cctggtaacg?cacaggattc?acgggacata?acgctgttga?aaaacaagaa?gaaggtcatc????780

aaaatgctga?ttatcgtggt?cattatcttt?ggactctgct?ggctgccact?gcagctctat????840

aatattctgt?atgtcacgat?accggaaatc?aacgactacc?acttcattag?catcgtctgg????900

ttttgctgcg?attggctggc?catgagcaat?agctgctaca?atccctttat?ttatggcatc????960

tacaatgaaa?aatttaagcg?ggaattcaac?aagcgatttg?cggcctgttt?ctgcaagttc???1020

aagacgagca?tggacgccca?cgaaaggacc?ttttcgatgc?acacccgcgc?cagctccata???1080

aggtcaacct?acgccaactc?ctcgatgcga?atccggagta?atctctttgg?tccggcgcgt???1140

ggtggtgtca?acaatgggaa?gccgggcttg?catatgccgc?gggtgcatgg?atccggtgct???1200

aacagcggca?tttacaacgg?aagtagtggg?cagaacaaca?atgtcaatgg?ccaacatcat???1260

cagcatcaaa?gcgtggttac?ctttgcggcc?actccgggtg?tttcggcacc?aggtgttggc???1320

gttgcaatgc?cgccgtggcg?gcgaaacaac?ttcaaacctc?tgcatccgaa?cgtaatcgaa???1380

tgcgaggacg?acgtggcact?catggagctg?ccatcaacca?cgccccccag?cgaggagttg???1440

gcatccgggg?ccggagtcca?gttggccctg?ctaagcaggg?agagctccag?ctgcatttgc???1500

gaacaggaat?ttggcagcca?aaccgaatgc?gatggcacct?gcatactcag?cgaggtgtcg???1560

cgagtccacc?tgcccggctc?gcaggcgaag?gacaaggatg?cgggcaagtc?cttgtggcaa???1620

ccacttta????????????????????????????????????????????????????????????1628

<210>18

<211>542

<212>PRT

＜213〉fruit bat (D.melanogaster)

<400>18

Met?Ala?Met?Asp?Leu?Ile?Glu?Gln?Glu?Ser?Arg?Leu?Glu?Phe?Leu?Pro

1???????????????5???????????????????10??????????????????15

Gly?Ala?Glu?Glu?Glu?Ala?Glu?Phe?Glu?Arg?Leu?Tyr?Ala?Ala?Pro?Ala

20??????????????????25??????????????????30

Glu?Ile?Val?Ala?Leu?Leu?Ser?Ile?Phe?Tyr?Gly?Gly?Ile?Ser?Ile?Val

35??????????????????40??????????????????45

Ala?Val?Ile?Gly?Asn?Thr?Leu?Val?Ile?Trp?Val?Val?Ala?Thr?Thr?Arg

50??????????????????55??????????????????60

Gln?Met?Arg?Thr?Val?Thr?Asn?Met?Tyr?Ile?Ala?Asn?Leu?Ala?Phe?Ala

65??????????????????70??????????????????75??????????????????80

Asp?Val?Ile?Ile?Gly?Leu?Phe?Cys?Ile?Pro?Phe?Gln?Phe?Gln?Ala?Ala

85??????????????????90??????????????????95

Leu?Leu?Gln?Ser?Trp?Asn?Leu?Pro?Trp?Phe?Met?Cys?Ser?Phe?Cys?Pro

100?????????????????105?????????????????110

Phe?Val?Gln?Ala?Leu?Ser?Val?Asn?Val?Ser?Val?Phe?Thr?Leu?Thr?Ala

115?????????????????120?????????????????125

Ile?Ala?Ile?Asp?Arg?His?Arg?Ala?Ile?Ile?Asn?Pro?Leu?Arg?Ala?Arg

130?????????????????135?????????????????140

Pro?Thr?Lys?Phe?Val?Ser?Lys?Phe?Ile?Ile?Gly?Gly?Ile?Trp?Met?Leu

145?????????????????150?????????????????155?????????????????160

Ala?Leu?Leu?Phe?Ala?Val?Pro?Phe?Ala?Ile?Ala?Phe?Arg?Val?Glu?Glu

165?????????????????170?????????????????175

Leu?Thr?Glu?Arg?Phe?Arg?Glu?Asn?Asn?Glu?Thr?Tyr?Asn?Val?Thr?Arg

180?????????????????185?????????????????190

Pro?Phe?Cys?Met?Asn?Lys?Asn?Leu?Ser?Asp?Asp?Gln?Leu?Gln?Ser?Phe

195?????????????????200?????????????????205

Arg?Tyr?Thr?Leu?Val?Phe?Val?Gln?Tyr?Leu?Val?Pro?Phe?Cys?Val?Ile

210?????????????????215?????????????????220

Ser?Phe?Val?Tyr?Ile?Gln?Met?Ala?Val?Arg?Leu?Trp?Gly?Thr?Arg?Ala

225?????????????????230?????????????????235?????????????????240

Pro?Gly?Asn?Ala?Gln?Asp?Ser?Arg?Asp?Ile?Thr?Leu?Leu?Lys?Asn?Lys

245?????????????????250?????????????????255

Lys?Lys?Val?Ile?Lys?Met?Leu?Ile?Ile?Val?Val?Ile?Ile?Phe?Gly?Leu

260?????????????????265?????????????????270

Cys?Trp?Leu?Pro?Leu?Gln?Leu?Tyr?Asn?Ile?Leu?Tyr?Val?Thr?Ile?Pro

275?????????????????280?????????????????285

Glu?Ile?Asn?Asp?Tyr?His?Phe?Ile?Ser?Ile?Val?Trp?Phe?Cys?Cys?Asp

290?????????????????295?????????????????300

Trp?Leu?Ala?Met?Ser?Asn?Ser?Cys?Tyr?Asn?Pro?Phe?Ile?Tyr?Gly?Ile

305?????????????????310?????????????????315?????????????????320

Tyr?Asn?Glu?Lys?Phe?Lys?Arg?Glu?Phe?Asn?Lys?Arg?Phe?Ala?Ala?Cys

325?????????????????330?????????????????335

Phe?Cys?Lys?Phe?Lys?Thr?Ser?Met?Asp?Ala?His?Glu?Arg?Thr?Phe?Ser

340?????????????????345?????????????????350

Met?His?Thr?Arg?Ala?Ser?Ser?Ile?Arg?Ser?Thr?Tyr?Ala?Asn?Ser?Ser

355?????????????????360?????????????????365

Met?Arg?Ile?Arg?Ser?Asn?Leu?Phe?Gly?Pro?Ala?Arg?Gly?Gly?Val?Asn

370?????????????????375?????????????????380

Asn?Gly?Lys?Pro?Gly?Leu?His?Met?Pro?Arg?Val?His?Gly?Ser?Gly?Ala

385?????????????????390?????????????????395?????????????????400

Asn?Ser?Gly?Ile?Tyr?Asn?Gly?Ser?Ser?Gly?Gln?Asn?Asn?Asn?Val?Asn

405?????????????????410?????????????????415

Gly?Gln?His?His?Gln?His?Gln?Ser?Val?Val?Thr?Phe?Ala?Ala?Thr?Pro

420?????????????????425?????????????????430

Gly?Val?Ser?Ala?Pro?Gly?Val?Gly?Val?Ala?Met?Pro?Pro?Trp?Arg?Arg

435?????????????????440?????????????????445

Asn?Asn?Phe?Lys?Pro?Leu?His?Pro?Asn?Val?Ile?Glu?Cys?Glu?Asp?Asp

450?????????????????455?????????????????460

Val?Ala?Leu?Met?Glu?Leu?Pro?Ser?Thr?Thr?Pro?Pro?Ser?Glu?Glu?Leu

465?????????????????470?????????????????475?????????????????480

Ala?Ser?Gly?Ala?Gly?Val?Gln?Leu?Ala?Leu?Leu?Ser?Arg?Glu?Ser?Ser

485?????????????????490?????????????????495

Ser?Cys?Ile?Cys?Glu?Gln?Glu?Phe?Gly?Ser?Gln?Thr?Glu?Cys?Asp?Gly

500?????????????????505?????????????????510

Thr?Cys?Ile?Leu?Ser?Glu?Val?Ser?Arg?Val?His?Leu?Pro?Gly?Ser?Gln

515?????????????????520?????????????????525

Ala?Lys?Asp?Lys?Asp?Ala?Gly?Lys?Ser?Leu?Trp?Gln?Pro?Leu

530?????????????????535?????????????????540

<210>19

<211>1451

<212>DNA

＜213〉fruit bat (D.melanogaster)

<400>19

atgtttacgt?ggctgatgat?ggatgtcctc?cagtttgtga?aaggggaaat?gacagccgat?????60

tcagaggcaa?atgccacaaa?ttggtataac?acgaacgaga?gcttatatac?cacggaactg????120

aaccatagat?ggattagtgg?tagttccaca?attcagccag?aggagtccct?ttatggcact????180

gatttgccca?cctatcaaca?ttgcatagcc?acgcggaatt?cctttgctga?cttgttcact????240

gtggtgctct?acggatttgt?gtgcattatc?ggattatttg?gcaacaccct?ggtgatctac????300

gtggtgttgc?gcttttccaa?aatgcaaacg?gtcacgaata?tatatatcct?gaatctggcg????360

gtggcagacg?agtgcttcct?gattggaata?ccctttctgc?tgtacacaat?gcgaatttgc????420

agctggcgat?tcggggagtt?tatgtgcaaa?gcctacatgg?tgagcacatc?catcacctcc????480

ttcacctcgt?cgatttttct?gctcatcatg?tccgcggatc?gatatatagc?ggtatgccac????540

ccgatttcct?cgccacgata?tcgaactctg?catattgcca?aagtggtctc?agcgattgcc????600

tggtcaactt?cagcggtcct?catgctgccc?gtgatccttt?atgccagcac?tgtggagcag????660

gaggatggca?tcaattactc?gtgcaacata?atgtggccag?atgcgtacaa?gaagcattcg????720

ggcaccacct?tcatactgta?cacatttttc?ctaggattcg?ccacaccgct?gtgctttatc????780

ctgagtttct?actacttggt?tataaggaaa?ctgcgatcgg?tgggtcccaa?accaggaacg????840

aagtccaagg?agaagaggcg?ggctcacagg?aaggtcactc?gactggtact?gacggtgata????900

agtgtataca?ttctatgttg?gctccctcac?tggatttctc?aggtggccct?gattcactcg????960

aatcccgcgc?aaagggacct?ctcccgactg?gaaatactca?ttttcctact?tctgggggca???1020

ctggtttact?cgaattcggc?ggtgaatccc?atactttatg?ccttcctaag?tgagaacttc???1080

cggaagagct?tcttcaaggc?ctttacctgt?atgaataagc?aggatatcaa?cgctcaactc???1140

cagctggagc?ccagtgtttt?caccaaacag?ggcagtaaaa?agaggggtgg?ctccaagcgc???1200

ctgttgacca?gcaatccgca?gattcctcca?ctgctgccac?tgaatgcggg?taacaacaat???1260

tcatcgacca?ccacatcctc?gaccacgaca?gcggaaaaga?ccggaaccac?ggggacacag???1320

aaatcatgca?attccaatgg?caaagtgaca?gctccgccgg?agaatttgat?tatatgtttg???1380

agcgagcagc?aggaggcatt?ttgcaccacc?gcgagaagag?gatcgggcgc?agtgcagcag???1440

acagatttgt?a????????????????????????????????????????????????????????1451

<210>20

<211>483

<212>PRT

＜213〉fruit bat (D.melanogaster)

<400>20

Met?Phe?Thr?Trp?Leu?Met?Met?Asp?Val?Leu?Gln?Phe?Val?Lys?Gly?Glu

1???????????????5???????????????????10??????????????????15

Met?Thr?Ala?Asp?Ser?Glu?Ala?Asn?Ala?Thr?Asn?Trp?Tyr?Asn?Thr?Asn

20??????????????????25??????????????????30

Glu?Ser?Leu?Tyr?Thr?Thr?Glu?Leu?Asn?His?Arg?Trp?Ile?Ser?Gly?Ser

35??????????????????40??????????????????45

Ser?Thr?Ile?Gln?Pro?Glu?Glu?Ser?Leu?Tyr?Gly?Thr?Asp?Leu?Pro?Thr

50??????????????????55??????????????????60

Tyr?Gln?His?Cys?Ile?Ala?Thr?Arg?Asn?Ser?Phe?Ala?Asp?Leu?Phe?Thr

65??????????????????70??????????????????75??????????????????80

Val?Val?Leu?Tyr?Gly?Phe?Val?Cys?Ile?Ile?Gly?Leu?Phe?Gly?Asn?Thr

85??????????????????90??????????????????95

Leu?Val?Ile?Tyr?Val?Val?Leu?Arg?Phe?Ser?Lys?Met?Gln?Thr?Val?Thr

100?????????????????105?????????????????110

Asn?Ile?Tyr?Ile?Leu?Asn?Leu?Ala?Val?Ala?Asp?Glu?Cys?Phe?Leu?Ile

115?????????????????120?????????????????125

Gly?Ile?Pro?Phe?Leu?Leu?Tyr?Thr?Met?Arg?Ile?Cys?Ser?Trp?Arg?Phe

130?????????????????135?????????????????140

Gly?Glu?Phe?Met?Cys?Lys?Ala?Tyr?Met?Val?Ser?Thr?Ser?Ile?Thr?Ser

145?????????????????150?????????????????155?????????????????160

Phe?Thr?Ser?Ser?Ile?Phe?Leu?Leu?Ile?Met?Ser?Ala?Asp?Arg?Tyr?Ile

165?????????????????170?????????????????175

Ala?Val?Cys?His?Pro?Ile?Ser?Ser?Pro?Arg?Tyr?Arg?Thr?Leu?His?Ile

180?????????????????185?????????????????190

Ala?Lys?Val?Val?Ser?Ala?Ile?Ala?Trp?Ser?Thr?Ser?Ala?Val?Leu?Met

195?????????????????200?????????????????205

Leu?Pro?Val?Ile?Leu?Tyr?Ala?Ser?Thr?Val?Glu?Gln?Glu?Asp?Gly?Ile

210?????????????????215?????????????????220

Asn?Tyr?Ser?Cys?Asn?Ile?Met?Trp?Pro?Asp?Ala?Tyr?Lys?Lys?His?Ser

225?????????????????230?????????????????235?????????????????240

Gly?Thr?Thr?Phe?Ile?Leu?Tyr?Thr?Phe?Phe?Leu?Gly?Phe?Ala?Thr?Pro

245?????????????????250?????????????????255

Leu?Cys?Phe?Ile?Leu?Ser?Phe?Tyr?Tyr?Leu?Val?Ile?Arg?Lys?Leu?Arg

260?????????????????265?????????????????270

Ser?Val?Gly?Pro?Lys?Pro?Gly?Thr?Lys?Ser?Lys?Glu?Lys?Arg?Arg?Ala

275?????????????????280?????????????????285

His?Arg?Lys?Val?Thr?Arg?Leu?Val?Leu?Thr?Val?Ile?Ser?Val?Tyr?Ile

290?????????????????295?????????????????300

Leu?Cys?Trp?Leu?Pro?His?Trp?Ile?Ser?Gln?Val?Ala?Leu?Ile?His?Ser

305?????????????????310?????????????????315?????????????????320

Asn?Pro?Ala?Gln?Arg?Asp?Leu?Ser?Arg?Leu?Glu?Ile?Leu?Ile?Phe?Leu

325?????????????????330?????????????????335

Leu?Leu?Gly?Ala?Leu?Val?Tyr?Ser?Asn?Ser?Ala?Val?Asn?Pro?Ile?Leu

340?????????????????345?????????????????350

Tyr?Ala?Phe?Leu?Ser?Glu?Asn?Phe?Arg?Lys?Ser?Phe?Phe?Lys?Ala?Phe

355?????????????????360?????????????????365

Thr?Cys?Met?Asn?Lys?Gln?Asp?Ile?Asn?Ala?Gln?Leu?Gln?Leu?Glu?Pro

370?????????????????375?????????????????380

Ser?Val?Phe?Thr?Lys?Gln?Gly?Ser?Lys?Lys?Arg?Gly?Gly?Ser?Lys?Arg

385?????????????????390?????????????????395?????????????????400

Leu?Leu?Thr?Ser?Asn?Pro?Gln?Ile?Pro?Pro?Leu?Leu?Pro?Leu?Asn?Ala

405?????????????????410?????????????????415

Gly?Asn?Asn?Asn?Ser?Ser?Thr?Thr?Thr?Ser?Ser?Thr?Thr?Thr?Ala?Glu

420?????????????????425?????????????????430

Lys?Thr?Gly?Thr?Thr?Gly?Thr?Gln?Lys?Ser?Cys?Asn?Ser?Asn?Gly?Lys

435?????????????????440?????????????????445

Val?Thr?Ala?Pro?Pro?Glu?Asn?Leu?Ile?Ile?Cys?Leu?Ser?Glu?Gln?Gln

450?????????????????455?????????????????460

Glu?Ala?Phe?Cys?Thr?Thr?Ala?Arg?Arg?Gly?Ser?Gly?Ala?Val?Gln?Gln

465?????????????????470?????????????????475?????????????????480

Thr?Asp?Leu

<210>21

<211>1754

<212>DNA

＜213〉fruit bat (D.melanogaster)

<400>21

atgttcaact?acgaggaggg?ggatgccgac?caggcggcca?tggctgcagc?ggctgcctat?????60

agggcactgc?tcgactacta?tgccaatgcg?ccaagtgcgg?cgggtcacat?agtgtcgctc????120

aacgtggcac?cctacaatgg?aactggaaac?ggaggcactg?tctccttggc?gggcaatgcg????180

acaagcagct?atggcgatga?tgatagggat?ggctatatgg?acaccgagcc?cagtgacctg????240

gtcaccgaac?tggccttctc?cctgggcacc?agttcaagtc?caagtcccag?ttccacaccc????300

gcttccagct?ccagtacttc?cactggcatg?cccgtctggc?tgatacccag?ctatagcatg????360

attctgctgt?tcgccgtgct?gggcaacctg?ctggtcatct?cgacgctggt?gcagaatcgc????420

cggatgcgta?ccataaccaa?cgtgttcctg?ctcaacctgg?ccatatcgga?catgctgctg????480

ggcgtgctct?gcatgcccgt?caccctggtg?ggcaccctgc?tgcgaaactt?catctttggc????540

gagttcctct?gcaagctctt?tcagttctcg?caagccgcct?ccgtggccgt?ttcgtcctgg????600

accttggtgg?ccatatcctg?tgagcgctac?tacgcgatat?gccatccact?gcgctcgcga????660

tcctggcaga?caatcagtca?cgcctacaag?atcatcggct?tcatctggct?gggcggcatc????720

ctctgcatga?cgcccatagc?ggtctttagt?caattgatac?ccaccagtcg?accgggctac?????780

tgcaagtgcc?gtgagttttg?gcccgaccag?ggatacgagc?tcttctacaa?catcctgctg?????840

gacttcctgc?tgctcgtcct?gccgcttctc?gtcctctgcg?tggcctacat?cctcatcacg?????900

cgtaccctgt?acgtaggcat?ggccaaggac?agcggacgca?tcctgcagca?atcgctgcct?????960

gtttccgcta?caacggccgg?cggaagcgca?ccgaatccgg?gcaccagcag?cagtagtaac????1020

tgcatcctgg?tcctgaccgc?caccgcagtc?tataatgaaa?atagtaacaa?taataatgga????1080

aattcagagg?gatccgcagg?cggaggatca?accaatatgg?caacgaccac?cttgacaacg????1140

agaccaacgg?ctccaactgt?gatcaccacc?accacgacga?ccacggtgac?gctggccaag????1200

acctcctcgc?ccagcattcg?cgtccacgat?gcggcacttc?gcaggtccaa?cgaggccaag????1260

accctggaga?gcaagaagcg?tgtggtcaag?atgctgttcg?tcctggtgct?ggagtttttc????1320

atctgctgga?ctccgctgta?cgtgatcaac?acgatggtca?tgctgatcgg?accggtggtg????1380

tacgagtatg?tcgactacac?ggccatcagt?ttcctccagc?tgctggccta?ctcatccagc????1440

tgctgcaatc?cgatcaccta?ctgcttcatg?aacgccagct?tccggcgcgc?ctttgtcgac????1500

accttcaagg?gtctgccctg?gcgtcgtgga?gcaggtgcca?gcggaggcgt?cggtggtgct????1560

gctggtggag?gactctccgc?cagccaggcg?ggcgcaggcc?cgggcgccta?tgcgagtgcc????1620

aacaccaaca?ttagtctcaa?tcccggccta?gccatgggta?tgggcacctg?gcggagtcgc????1680

tcacgccacg?agtttctcaa?tgcggtggtg?accaccaata?gtgccgccgc?cgccgtcaac????1740

agtcctcagc?tcta??????????????????????????????????????????????????????1754

<210>22

<211>584

<212>PRT

＜213〉fruit bat (D.melanogaster)

<400>22

Met?Phe?Asn?Tyr?Glu?Glu?Gly?Asp?Ala?Asp?Gln?Ala?Ala?Met?Ala?Ala

1???????????????5???????????????????10??????????????????15

Ala?Ala?Ala?Tyr?Arg?Ala?Leu?Leu?Asp?Tyr?Tyr?Ala?Asn?Ala?Pro?Ser

20??????????????????25??????????????????30

Ala?Ala?Gly?His?Ile?Val?Ser?Leu?Asn?Val?Ala?Pro?Tyr?Asn?Gly?Thr

35??????????????????40??????????????????45

Gly?Asn?Gly?Gly?Thr?Val?Ser?Leu?Ala?Gly?Asn?Ala?Thr?Ser?Ser?Tyr

50??????????????????55??????????????????60

Gly?Asp?Asp?Asp?Arg?Asp?Gly?Tyr?Met?Asp?Thr?Glu?Pro?Ser?Asp?Leu

65??????????????????70??????????????????75??????????????????80

Val?Thr?Glu?Leu?Ala?Phe?Ser?Leu?Gly?Thr?Ser?Ser?Ser?Pro?Ser?Pro

85??????????????????90??????????????????95

Ser?Ser?Thr?Pro?Ala?Ser?Ser?Ser?Ser?Thr?Ser?Thr?Gly?Met?Pro?Val

100?????????????????105?????????????????110

Trp?Leu?Ile?Pro?Ser?Tyr?Ser?Met?Ile?Leu?Leu?Phe?Ala?Val?Leu?Gly

115?????????????????120?????????????????125

Asn?Leu?Leu?Val?Ile?Ser?Thr?Leu?Val?Gln?Asn?Arg?Arg?Met?Arg?Thr

130?????????????????135?????????????????140

Ile?Thr?Asn?Val?Phe?Leu?Leu?Asn?Leu?Ala?Ile?Ser?Asp?Met?Leu?Leu

145?????????????????150?????????????????155?????????????????160

Gly?Val?Leu?Cys?Met?Pro?Val?Thr?Leu?Val?Gly?Thr?Leu?Leu?Arg?Asn

165?????????????????170?????????????????175

Phe?Ile?Phe?Gly?Glu?Phe?Leu?Cys?Lys?Leu?Phe?Gln?Phe?Ser?Gln?Ala

180?????????????????185?????????????????190

Ala?Ser?Val?Ala?Val?Ser?Ser?Trp?Thr?Leu?Val?Ala?Ile?Ser?Cys?Glu

195?????????????????200?????????????????205

Arg?Tyr?Tyr?Ala?Ile?Cys?His?Pro?Leu?Arg?Ser?Arg?Ser?Trp?Gln?Thr

210?????????????????215?????????????????220

Ile?Ser?His?Ala?Tyr?Lys?Ile?Ile?Gly?Phe?Ile?Trp?Leu?Gly?Gly?Ile

225?????????????????230?????????????????235?????????????????240

Leu?Cys?Met?Thr?Pro?Ile?Ala?Val?Phe?Ser?Gln?Leu?Ile?Pro?Thr?Ser

245?????????????????250?????????????????255

Arg?Pro?Gly?Tyr?Cys?Lys?Cys?Arg?Glu?Phe?Trp?Pro?Asp?Gln?Gly?Tyr

260?????????????????265?????????????????270

Glu?Leu?Phe?Tyr?Asn?Ile?Leu?Leu?Asp?Phe?Leu?Leu?Leu?Val?Leu?Pro

275?????????????????280?????????????????285

Leu?Leu?Val?Leu?Cys?Val?Ala?Tyr?Ile?Leu?Ile?Thr?Arg?Thr?Leu?Tyr

290?????????????????295?????????????????300

Val?Gly?Met?Ala?Lys?Asp?Ser?Gly?Arg?Ile?Leu?Gln?Gln?Ser?Leu?Pro

305?????????????????310?????????????????315?????????????????320

Val?Ser?Ala?Thr?Thr?Ala?Gly?Gly?Ser?Ala?Pro?Asn?Pro?Gly?Thr?Ser

325?????????????????330?????????????????335

Ser?Ser?Ser?Asn?Cys?Ile?Leu?Val?Leu?Thr?Ala?Thr?Ala?Val?Tyr?Asn

340?????????????????345?????????????????350

Glu?Asn?Ser?Asn?Asn?Asn?Asn?Gly?Asn?Ser?Glu?Gly?Ser?Ala?Gly?Gly

355?????????????????360?????????????????365

Gly?Ser?Thr?Asn?Met?Ala?Thr?Thr?Thr?Leu?Thr?Thr?Arg?Pro?Thr?Ala

370?????????????????375?????????????????380

Pro?Thr?Val?Ile?Thr?Thr?Thr?Thr?Thr?Thr?Thr?Val?Thr?Leu?Ala?Lys

385?????????????????390?????????????????395?????????????????400

Thr?Ser?Ser?Pro?Ser?Ile?Arg?Val?His?Asp?Ala?Ala?Leu?Arg?Arg?Ser

405?????????????????410?????????????????415

Asn?Glu?Ala?Lys?Thr?Leu?Glu?Ser?Lys?Lys?Arg?Val?Val?Lys?Met?Leu

420?????????????????425?????????????????430

Phe?Val?Leu?Val?Leu?Glu?Phe?Phe?Ile?Cys?Trp?Thr?Pro?Leu?Tyr?Val

435?????????????????440?????????????????445

Ile?Asn?Thr?Met?Val?Met?Leu?Ile?Gly?Pro?Val?Val?Tyr?Glu?Tyr?Val

450?????????????????455?????????????????460

Asp?Tyr?Thr?Ala?Ile?Ser?Phe?Leu?Gln?Leu?Leu?Ala?Tyr?Ser?Ser?Ser

465?????????????????470?????????????????475?????????????????480

Cys?Cys?Asn?Pro?Ile?Thr?Tyr?Cys?Phe?Met?Asn?Ala?Ser?Phe?Arg?Arg

485?????????????????490?????????????????495

Ala?Phe?Val?Asp?Thr?Phe?Lys?Gly?Leu?Pro?Trp?Arg?Arg?Gly?Ala?Gly

500?????????????????505?????????????????510

Ala?Ser?Gly?Gly?Val?Gly?Gly?Ala?Ala?Gly?Gly?Gly?Leu?Ser?Ala?Ser

515?????????????????520?????????????????525

Gln?Ala?Gly?Ala?Gly?Pro?Gly?Ala?Tyr?Ala?Ser?Ala?Asn?Thr?Asn?Ile

530?????????????????535?????????????????540

Ser?Leu?Asn?Pro?Gly?Leu?Ala?Met?Gly?Met?Gly?Thr?Trp?Arg?Ser?Arg

545?????????????????550?????????????????555?????????????????560

Ser?Arg?His?Glu?Phe?Leu?Asn?Ala?Val?Val?Thr?Thr?Asn?Ser?Ala?Ala

565?????????????????570?????????????????575

Ala?Ala?Val?Asn?Ser?Pro?Gln?Leu

580

<210>23

<211>1452

<212>DNA

＜213〉fruit bat (D.melanogaster)

<400>23

atgtacgcct?ccttgatgga?cgttggccag?acgttggcag?ccaggctggc?ggatagcgac?????60

ggcaacgggg?ccaatgacag?cggactcctg?gcaaccggac?aaggtctgga?gcaggagcag????120

gagggtctgg?cactggatat?gggccacaat?gccagcgccg?acggcggaat?agtaccgtat????180

gtgcccgtgc?tggaccgccc?ggagacgtac?attgtcaccg?tgctgtacac?gctcatcttc????240

attgtgggag?ttttgggcaa?cggcgcgctg?gtcatcatct?tctttcgcca?ccgctccatg????300

cgcaacatac?ccaacacata?cattctttca?ctggccctgg?ctgatctgtt?ggttatattg????360

gtgtgtgtac?ctgtggccac?gattgtctac?acgcaggaaa?gctggccctt?tgagcggaac????420

atgtgccgca?tcagcgagtt?ctttaaggac?atatccatcg?gggtgtccgt?gtttacactg????480

accgcccttt?ccggcgagcg?gtactgcgcc?attgtaaatc?ccctacgcaa?gcttcagacc????540

aagccgctca?ctgtctttac?tgcggtgatg?atctggatcc?tggccatcct?actgggcatg????600

ccttcggttc?ttttctccga?catcaagtcc?taccctgtgt?tcacagccac?cggtaacatg????660

accattgaag?tgtgctcccc?atttcgcgac?ccggagtatg?caaagttcat?ggtggcgggc????720

aaggcactgg?tgtactacct?gttgccgctg?tccatcattg?gggcgctata?catcatgatg????780

gccaagcggc?tccatatgag?cgcccgcaac?atgcccggcg?aacagcagag?catgcagagc????840

cgcacccagg?ctagggcccg?actccatgtg?gcgcgcatgg?tggtagcatt?cgtggtggtg????900

ttcttcatct?gcttcttccc?gtaccacgtg?tttgagctgt?ggtaccactt?ctacccaacg????960

gctgaggagg?acttcgatga?gttctggaac?gtgctgcgca?tccttcctaa?actcgtgcgt???1020

caaccccgtg?gcctctactg?cgtgtccggg?gtgtttcggc?agcactttaa?tcgctacctc???1080

tgctgcatct?gcgtcaagcg?gcagccgcac?ctgcggcagc?actcaacggc?cactggaatg???1140

atggacaata?ccagtgtgat?gtccatgcgc?cgctccacgt?acgtgggtgg?aaccgctggc???1200

aatctgcggg?cctcgctgca?ccggaacagc?aatcacggag?ttggtggagc?tggaggtgga???1260

gtaggaggag?gagtagggtc?aggtcgtgtg?ggcagctttc?atcggcagga?ctcgatgccc???1320

ctgcagcacg?gaaatgccca?cggaggtggt?gcgggcgggg?gatcctccgg?acttggagcc???1380

ggcgggcgga?cggcggcagt?gagcgaaaag?agctttataa?atcgttacga?aagtggcgta???1440

atgcgctact?aa???????????????????????????????????????????????????????1452

<210>24

<211>483

<212>PRT

＜213〉fruit bat (D.melanogaster)

<400>24

Met?Tyr?Ala?Ser?Leu?Met?Asp?Val?Gly?Gln?Thr?Leu?Ala?Ala?Arg?Leu

1???????????????5???????????????????10??????????????????15

Ala?Asp?Ser?Asp?Gly?Asn?Gly?Ala?Asn?Asp?Ser?Gly?Leu?Leu?Ala?Thr

20??????????????????25??????????????????30

Gly?Gln?Gly?Leu?Glu?Gln?Glu?Gln?Glu?Gly?Leu?Ala?Leu?Asp?Met?Gly

35??????????????????40??????????????????45

His?Asn?Ala?Ser?Ala?Asp?Gly?Gly?Ile?Val?Pro?Tyr?Val?Pro?Val?Leu

50??????????????????55??????????????????60

Asp?Arg?Pro?Glu?Thr?Tyr?Ile?Val?Thr?Val?Leu?Tyr?Thr?Leu?Ile?Phe

65??????????????????70??????????????????75??????????????????80

Ile?Val?Gly?Val?Leu?Gly?Asn?Gly?Thr?Leu?Val?Ile?Ile?Phe?Phe?Arg

85??????????????????90??????????????????95

His?Arg?Ser?Met?Arg?Asn?Ile?Pro?Asn?Thr?Tyr?Ile?Leu?Ser?Leu?Ala

100?????????????????105?????????????????110

Leu?Ala?Asp?Leu?Leu?Val?Ile?Leu?Val?Cys?Val?Pro?Val?Ala?Thr?Ile

115?????????????????120?????????????????125

Val?Tyr?Thr?Gln?Glu?Ser?Trp?Pro?Phe?Glu?Arg?Asn?Met?Cys?Arg?Ile

130?????????????????135?????????????????140

Ser?Glu?Phe?Phe?Lys?Asp?Ile?Ser?Ile?Gly?Val?Ser?Val?Phe?Thr?Leu

145?????????????????150?????????????????155?????????????????160

Thr?Ala?Leu?Ser?Gly?Glu?Arg?Tyr?Cys?Ala?Ile?Val?Asn?Pro?Leu?Arg

165?????????????????170?????????????????175

Lys?Leu?Gln?Thr?Lys?Pro?Leu?Thr?Val?Phe?Thr?Ala?Val?Met?Ile?Trp

180?????????????????185?????????????????190

Ile?Leu?Ala?Ile?Leu?Leu?Gly?Met?Pro?Ser?Val?Leu?Phe?Ser?Asp?Ile

195?????????????????200?????????????????205

Lys?Ser?Tyr?Pro?Val?Phe?Thr?Ala?Thr?Gly?Asn?Met?Thr?Ile?Glu?Val

210?????????????????215?????????????????220

Cys?Ser?Pro?Phe?Arg?Asp?Pro?Glu?Tyr?Ala?Lys?Phe?Met?Val?Ala?Gly

225?????????????????230?????????????????235?????????????????240

Lys?Ala?Leu?Val?Tyr?Tyr?Leu?Leu?Pro?Leu?Ser?Ile?Ile?Gly?Ala?Leu

245?????????????????250?????????????????255

Tyr?Ile?Met?Met?Ala?Lys?Arg?Leu?His?Met?Ser?Ala?Arg?Asn?Met?Pro

260?????????????????265?????????????????270

Gly?Glu?Gln?Gln?Ser?Met?Gln?Ser?Arg?Thr?Gln?Ala?Arg?Ala?Arg?Leu

275?????????????????280?????????????????285

His?Val?Ala?Arg?Met?Val?Val?Ala?Phe?Val?Val?Val?Phe?Phe?Ile?Cys

290?????????????????295?????????????????300

Phe?Phe?Pro?Tyr?His?Val?Phe?Glu?Leu?Trp?Tyr?His?Phe?Tyr?Pro?Thr

305?????????????????310?????????????????315?????????????????320

Ala?Glu?Glu?Asp?Phe?Asp?Glu?Phe?Trp?Asn?Val?Leu?Arg?Ile?Leu?Pro

325?????????????????330?????????????????335

Lys?Leu?Val?Arg?Gln?Pro?Arg?Gly?Leu?Tyr?Cys?Val?Ser?Gly?Val?Phe

340?????????????????345?????????????????350

Arg?Gln?His?Phe?Asn?Arg?Tyr?Leu?Cys?Cys?Ile?Cys?Val?Lys?Arg?Gln

355?????????????????360?????????????????365

Pro?His?Leu?Arg?Gln?His?Ser?Thr?Ala?Thr?Gly?Met?Met?Asp?Asn?Thr

370?????????????????375?????????????????380

Ser?Val?Met?Ser?Met?Arg?Arg?Ser?Thr?Tyr?Val?Gly?Gly?Thr?Ala?Gly

385?????????????????390?????????????????395?????????????????400

Asn?Leu?Arg?Ala?Ser?Leu?His?Arg?Asn?Ser?Asn?His?Gly?Val?Gly?Gly

405?????????????????410?????????????????415

Ala?Gly?Gly?Gly?Val?Gly?Gly?Gly?Val?Gly?Ser?Gly?Arg?Val?Gly?Ser

420?????????????????425?????????????????430

Phe?His?Arg?Gln?Asp?Ser?Met?Pro?Leu?Gln?His?Gly?Asn?Ala?His?Gly

435?????????????????440?????????????????445

Gly?Gly?Ala?Gly?Gly?Gly?Ser?Ser?Gly?Leu?Gly?Ala?Gly?Gly?Arg?Thr

450?????????????????455?????????????????460

Ala?Ala?Val?Ser?Glu?Lys?Ser?Phe?Ile?Asn?Arg?Tyr?Glu?Ser?Gly?Val

465?????????????????470?????????????????475?????????????????480

Met?Arg?Tyr

<210>25

<211>10

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>25

Thr?Asp?Val?Asp?His?Val?Phe?Leu?Arg?Phe

1???????????????5???????????????????10

<210>26

<211>9

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>26

Asp?Pro?Lys?Gln?Asp?Phe?Met?Arg?Phe

1???????????????5

<210>27

<211>7

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>27

Pro?Asp?Asn?Phe?Met?Arg?Phe

1???????????????5

<210>28

<211>9

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>28

Thr?Pro?Ala?Glu?Asp?Phe?Met?Arg?Phe

1???????????????5

<210>29

<211>9

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>29

Ser?Leu?Lys?Gln?Asp?Phe?Met?His?Phe

1???????????????5

<210>30

<211>9

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>30

Ser?Val?Lys?Gln?Asp?Phe?Met?His?Phe

1???????????????5

<210>31

<211>6

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>31

Ala?Ala?Met?Asp?Arg?Tyr

1???????????????5

<210>32

<211>9

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>32

Ser?Val?Gln?Asp?Asn?Phe?Met?His?Phe

1???????????????5

<210>33

<211>10

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>33

Ala?Arg?Gly?Pro?Gln?Leu?Arg?Leu?Arg?Phe

1???????????????5???????????????????10

<210>34

<211>10

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>34

Gly?Asp?Gly?Arg?Leu?Tyr?Ala?Phe?Gly?Leu

1???????????????5???????????????????10

<210>35

<211>8

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>35

Asp?Arg?Leu?Tyr?Ser?Phe?Gly?Leu

1???????????????5

<210>36

<211>13

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>36

Ala?Pro?Ser?Gly?Ala?Gln?Arg?Leu?Tyr?Gly?Phe?Gly?Leu

1???????????????5???????????????????10

<210>37

<211>9

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>37

Gly?Gly?Ser?Leu?Tyr?Ser?Phe?Gly?Leu

1???????????????5

<210>38

<211>4

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>38

Phe?Ile?Arg?Phe

1

<210>39

<211>7

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>39

Lys?Asn?Glu?Phe?Ile?Arg?Phe

1???????????????5

<210>40

<211>4

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>40

Phe?Met?Arg?Phe

1

<210>41

<211>7

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>41

Lys?Ser?Ala?Phe?Met?Arg?Phe

1???????????????5

<210>42

<211>7

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>42

Lys?Pro?Asn?Phe?Leu?Arg?Phe

1???????????????5

<210>43

<211>4

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>43

Phe?Leu?Arg?Phe

1

<210>44

<211>4

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>44

Tyr?Leu?Arg?Phe

1

<210>45

<211>7

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>45

Lys?Pro?Asn?Phe?Leu?Arg?Tyr

1??????????????5

<210>46

<211>8

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>46

Thr?Asn?Arg?Asn?Phe?Leu?Arg?Phe

1???????????????5

<210>47

<211>9

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>47

Arg?Asn?Lys?Phe?Glu?Phe?Ile?Arg?Phe

1???????????????5

<210>48

<211>8

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>48

Ala?Gly?Pro?Arg?Phe?Ile?Arg?Phe

1???????????????5

<210>49

<211>9

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>49

Gly?Leu?Gly?Pro?Arg?Pro?Leu?Arg?Phe

1???????????????5

<210>50

<211>2

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>50

Ile?Leu

1

<210>51

<211>8

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>51

Ala?Gly?Ala?Lys?Ile?Phe?Arg?Phe

1???????????????5

<210>52

<211>9

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>52

Ala?Pro?Lys?Pro?Lys?Phe?Ile?Arg?Phe

1???????????????5

<210>53

<211>8

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>53

Lys?Ser?Ala?Phe?Val?Leu?Arg?Phe

1???????????????5

<210>54

<211>9

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>54

Thr?Lys?Phe?Gln?Asp?Phe?Leu?Arg?Phe

1???????????????5

<210>55

<211>10

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>55

Ser?Ala?Glu?Pro?Phe?Gly?Thr?Met?Arg?Phe

1???????????????5???????????????????10

<210>56

<211>12

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>56

Ala?Ser?Glu?Asp?Ala?Leu?Phe?Gly?Thr?Met?Arg?Phe

1???????????????5???????????????????10

<210>57

<211>13

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>57

Ser?Ala?Asp?Asp?Ser?Ala?Pro?Phe?Gly?Thr?Met?Arg?Phe

1???????????????5???????????????????10

<210>58

<211>12

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>58

Glu?Asp?Gly?Asn?Ala?Pro?Phe?Gly?Thr?Met?Arg?Phe

1???????????????5???????????????????10

<210>59

<211>8

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>59

Phe?Leu?Phe?Gln?Pro?Gln?Arg?Phe

1???????????????5

<210>60

<211>9

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>60

Ser?Ala?Asp?Pro?Asn?Phe?Leu?Arg?Phe

1???????????????5

<210>61

<211>8

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>61

Ser?Gln?Pro?Asn?Phe?Leu?Arg?Phe

1???????????????5

<210>62

<211>10

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>62

Ala?Ser?Gly?Asp?Pro?Asn?Phe?Leu?Arg?Phe

1???????????????5???????????????????10

<210>63

<211>8

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>63

Ser?Asp?Pro?Asn?Phe?Leu?Arg?Phe

1???????????????5

<210>64

<211>10

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>64

Ala?Ala?Ala?Asp?Pro?Asn?Phe?Leu?Arg?Phe

1???????????????5???????????????????10

<210>65

<211>6

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>65

Pro?Asn?Phe?Leu?Arg?Phe

1???????????????5

<210>66

<211>6

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>66

Lys?Pro?Phe?Leu?Arg?Phe

1???????????????5

<210>67

<211>10

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>67

Ala?Gly?Ser?Asp?Pro?Asn?Phe?Leu?Arg?Phe

1???????????????5???????????????????10

<210>68

<211>7

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>68

Lys?Pro?Asn?Phe?Leu?Arg?Tyr

1???????????????5

<210>69

<211>8

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>69

Ser?Pro?Arg?Glu?Pro?Ile?Arg?Phe

1???????????????5

<210>70

<211>8

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>70

Leu?Arg?Gly?Glu?Pro?Ile?Arg?Phe

1???????????????5

<210>71

<211>8

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>71

Ser?Ala?Asp?Asp?Ser?Ala?Pro?Phe?Gly?Thr?Met?Arg?Phe

1???????????????5???????????????????10

<210>77

<211>7

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>77

Lys?Pro?Thr?Phe?Ile?Arg?Phe

1???????????????5

<210>78

<211>8

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>78

Ala?Ser?Pro?Ser?Phe?Ile?Arg?Phe

1???????????????5

<210>79

<211>7

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>79

Gly?Ala?Lys?Phe?Ile?Arg?Phe

1???????????????5

<210>80

<211>8

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>80

Ala?Gly?Ala?Lys?Phe?Ile?Arg?Phe

1???????????????5

<210>81

<211>9

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>81

Ser?Pro?Leu?Gly?Thr?Met?Arg?Phe

1???????????????5

<210>72

<211>11

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>72

Glu?Ala?Glu?Glu?Pro?Leu?Gly?Thr?Met?Arg?Phe

1???????????????5???????????????????10

<210>73

<211>12

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>73

Ala?Ser?Glu?Asp?Ala?Leu?Phe?Gly?Thr?Met?Arg?Phe

1???????????????5???????????????????10

<210>74

<211>12

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>74

Glu?Asp?Gly?Asn?Ala?Pro?Phe?Gly?Thr?Met?Arg?Phe

1???????????????5???????????????????10

<210>75

<211>10

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>75

Ser?Ala?Glu?Pro?Phe?Gly?Thr?Met?Arg?Phe

1???????????????5???????????????????10

<210>76

<211>13

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>76

Ala?Pro?Lys?Pro?Lys?Phe?Ile?Arg?Phe

1???????????????5

<210>82

<211>7

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>82

Lys?Ser?Ala?Tyr?Met?Arg?Phe

1???????????????5

<210>83

<211>11

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>83

Ser?Pro?Met?Gln?Arg?Ser?Ser?Met?Val?Arg?Phe

1???????????????5???????????????????10

<210>84

<211>11

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>84

Ser?Pro?Met?Glu?Arg?Ser?Ala?Met?Val?Arg?Phe

1???????????????5???????????????????10

<210>85

<211>11

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>85

Ser?Pro?Met?Asp?Arg?Ser?Lys?Met?Val?Arg?Phe

1???????????????5???????????????????10

<210>86

<211>7

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>86

Lys?Asn?Glu?Phe?Ile?Arg?Phe

1???????????????5

<210>87

<211>7

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>87

Lys?Pro?Ser?Phe?Val?Arg?Phe

1???????????????5

<210>88

<211>11

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>88

Gln?Pro?Lys?Ala?Arg?Ser?Gly?Tyr?Ile?Arg?Phe

1???????????????5???????????????????10

<210>89

<211>9

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>89

Ala?Met?Arg?Asn?Ala?Leu?Val?Arg?Phe

1???????????????5

<210>90

<211>12

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>90

Ala?Ser?Gly?Gly?Met?Arg?Asn?Ala?Leu?Val?Arg?Phe

1???????????????5???????????????????10

<210>91

<211>10

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>91

Asn?Gly?Ala?Pro?Gln?Pro?Phe?Val?Arg?Phe

1???????????????5???????????????????10

<210>92

<211>9

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>92

Arg?Asn?Lys?Phe?Glu?Phe?Ile?Arg?Phe

1???????????????5

<210>93

<211>14

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>93

Ser?Asp?Arg?Pro?Thr?Arg?Ala?Met?Asp?Ser?Pro?Ile?Arg?Phe

1???????????????5???????????????????10

<210>94

<211>10

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>94

Ala?Ala?Asp?Gly?Ala?Pro?Leu?Ile?Arg?Phe

1???????????????5???????????????????10

<210>95

<211>10

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>95

Ala?Pro?Glu?Ala?Ser?Pro?Phe?Ile?Arg?Phe

1???????????????5???????????????????10

<210>96

<211>10

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>96

Ala?Ser?Pro?Ser?Ala?Pro?Leu?Ile?Arg?Phe

1???????????????5???????????????????10

<210>97

<211>10

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>97

Ser?Pro?Ser?Ala?Val?Pro?Leu?Ile?Arg?Phe

1???????????????5???????????????????10

<210>98

<211>9

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>98

Ala?Ser?Ser?Ala?Pro?Leu?Ile?Arg?Phe

1???????????????5

<210>99

<211>7

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>99

Lys?His?Glu?Tyr?Leu?Arg?Phe

1???????????????5

<210>100

<211>6

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>100

Ser?Leu?Asp?Tyr?Arg?Phe

1???????????????5

<210>101

<211>14

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>101

Glu?Ile?Val?Phe?His?Gln?Ile?Ser?Pro?Ile?Phe?Phe?Arg?Phe

1???????????????5???????????????????10

<210>102

<211>9

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>102

Gly?Gly?Pro?Gln?Gly?Pro?Leu?Arg?Phe

1???????????????5

<210>103

<211>8

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>103

Gly?Pro?Ser?Gly?Pro?Leu?Arg?Phe

1???????????????5

<210>104

<211>7

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>104

Ala?Gln?Thr?Phe?Val?Arg?Phe

1???????????????5

<210>105

<211>7

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>105

Gly?Gln?Thr?Phe?Val?Arg?Phe

1???????????????5

<210>106

<211>7

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>106

Lys?Ser?Ala?Phe?Val?Arg?Phe

1???????????????5

<210>107

<211>7

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>107

Lys?Ser?Gln?Tyr?Ile?Arg?Phe

1???????????????5

<210>108

<211>8

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>108

Asp?Val?Pro?Gly?Val?Leu?Arg?Phe

1???????????????5

<210>109

<211>9

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>109

Lys?Ser?Val?Pro?Gly?Val?Leu?Arg?Phe

1???????????????5

<210>110

<211>9

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>110

Ser?Glu?Val?Pro?Gly?Val?Leu?Arg?Phe

1???????????????5

<210>111

<211>8

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>111

Ser?Val?Pro?Gly?Val?Leu?Arg?Phe

1???????????????5

<210>112

<211>12

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>112

Asp?Phe?Asp?Gly?Ala?Met?Pro?Gly?Val?Leu?Arg?Phe

1???????????????5???????????????????10

<210>113

<211>8

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>113

Glu?Ile?Pro?Gly?Val?Leu?Arg?Phe

1???????????????5

<210>114

<211>7

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>114

Trp?Ala?Asn?Gln?Val?Arg?Phe

1???????????????5

<210>115

<211>9

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>115

Ala?Ser?Trp?Ala?Ser?Ser?Val?Arg?Phe

1???????????????5

<210>116

<211>5

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>116

Ala?Met?Met?Arg?Phe

1???????????????5

<210>117

<211>9

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>117

Gly?Leu?Gly?Pro?Arg?Pro?Leu?Arg?Phe

1???????????????5

<210>118

<211>9

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>118

Ser?Pro?Ser?Ala?Lys?Trp?Met?Arg?Phe

1???????????????5

<210>119

<211>9

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>119

Thr?Lys?Phe?Gln?Asp?Phe?Leu?Arg?Phe

1???????????????5

<210>120

<211>10

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>120

Glu?Asp?Arg?Asp?Tyr?Arg?Pro?Leu?Gln?Phe

1???????????????5???????????????????10

<210>121

<211>4

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>121

Phe?Ile?Arg?Phe

1

<210>122

<211>8

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>122

Ala?Val?Pro?Gly?Val?Leu?Arg?Phe

1???????????????5

<210>123

<211>9

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>123

Gly?Asp?Val?Pro?Gly?Val?Leu?Arg?Phe

1???????????????5

<210>124

<211>13

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>124

Ser?Asp?Ile?Gly?Ile?Ser?Glu?Pro?Asn?Phe?Leu?Arg?Phe

1???????????????5???????????????????10

<210>125

<211>9

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>125

Ser?Gly?Lys?Pro?Thr?Phe?Ile?Arg?Phe

1???????????????5

<210>126

<211>11

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>126

Ala?Glu?Gly?Leu?Ser?Ser?Pro?Leu?Ile?Arg?Phe

1???????????????5???????????????????10

<210>127

<211>8

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>127

Phe?Asp?Arg?Asp?Phe?Met?Arg?Phe

1???????????????5

<210>128

<211>8

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>128

Ala?Gly?Pro?Arg?Phe?Ile?Arg?Phe

1???????????????5

<210>129

<211>8

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>129

Gly?Met?Pro?Gly?Val?Leu?Arg?Phe

1???????????????5

<210>130

<211>2

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>130

Ile?Leu

1

<210>131

<211>8

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>131

Leu?Gln?Pro?Asn?Phe?Leu?Arg?Phe

1???????????????5

<210>132

<211>7

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>132

Lys?Pro?Asn?Phe?Ile?Arg?Phe

1???????????????5

<210>133

<211>4

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>133

Phe?Met?Arg?Phe

1

<210>134

<211>4

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>134

Phe?Leu?Arg?Phe

1

<210>135

<211>4

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>135

Tyr?Ile?Arg?Phe

1

<210>136

<211>7

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>136

Gly?Asn?Ser?Phe?Leu?Arg?Phe

1???????????????5

<210>137

<211>7

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>137

Asp?Pro?Ser?Phe?Leu?Arg?Phe

1???????????????5

<210>138

<211>6

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>138

Gln?Asp?Phe?Met?Arg?Phe

1???????????????5

<210>139

<211>9

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>139

Lys?Pro?Asn?Gln?Asp?Phe?Met?Arg?Phe

1???????????????5

<210>140

<211>10

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>140

Thr?Asp?Val?Asp?His?Val?Phe?Leu?Arg?Phe

1???????????????5???????????????????10

<210>141

<211>6

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>141

Ala?Ala?Met?Asp?Arg?Tyr

1???????????????5

<210>142

<211>9

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>142

Ser?Pro?Lys?Gln?Asp?Phe?Met?Arg?Phe

1???????????????5

<210>143

<211>7

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>143

Pro?Asp?Asn?Phe?Met?Arg?Phe

1???????????????5

<210>144

<211>9

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>144

Asp?Pro?Lys?Gln?Asp?Phe?Met?Arg?Phe

1???????????????5

<210>145

<211>9

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>145

Thr?Pro?Ala?Glu?Asp?Phe?Met?Arg?Phe

1???????????????5

<210>146

<211>7

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>146

Ser?Asp?Asn?Phe?Met?Arg?Phe

1???????????????5

<210>147

<211>4

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>147

Tyr?Leu?Arg?Phe

1

<210>148

<211>8

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>148

Ser?Asp?Arg?Asn?Phe?Leu?Arg?Phe

1???????????????5

<210>149

<211>8

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>149

Thr?Asn?Arg?Asn?Phe?Leu?Arg?Phe

1???????????????5

<210>150

<211>10

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>150

Pro?Asp?Val?Asp?His?Val?Phe?Leu?Arg?Phe

1???????????????5???????????????????10

<210>151

<211>10

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>151

Gln?Asp?Val?Asp?His?Val?Phe?Leu?Arg?Phe

1???????????????5???????????????????10

<210>152

<211>8

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>152

Phe?Leu?Phe?Gln?Pro?Gln?Arg?Phe

1???????????????5

<210>153

<211>10

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>153

Ala?Arg?Gly?Pro?Gln?Leu?Arg?Leu?Arg?Phe

1???????????????5???????????????????10

<210>154

<211>9

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>154

Phe?Asp?Asp?Tyr?Gly?His?Leu?Arg?Phe

1???????????????5

<210>155

<211>9

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>155

Phe?Asp?Asp?Tyr?Gly?His?Leu?Arg?Phe

1???????????????5

<210>156

<211>8

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>156

Met?Asp?Ser?Asn?Phe?Ile?Arg?Phe

1???????????????5

<210>157

<211>9

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>157

Phe?Asp?Asp?Tyr?Gly?His?Leu?Arg?Phe

1???????????????5

<210>158

<211>9

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>158

Phe?Asp?Asp?Tyr?Gly?His?Leu?Arg?Phe

1???????????????5

<210>159

<211>9

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>159

Phe?Asp?Asp?Tyr?Gly?His?Met?Arg?Phe

1???????????????5

<210>160

<211>14

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>160

Gly?Gly?Asp?Asp?Gln?Phe?Asp?Asp?Tyr?Gly?His?Met?Arg?Phe

1???????????????5???????????????????10

<210>161

<211>8

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>161

Ser?Arg?Pro?Tyr?Ser?Phe?Gly?Leu

1???????????????5

<210>162

<211>7

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>162

Asp?Tyr?Gly?His?Met?Arg?Phe

1???????????????5

<210>163

<211>9

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>163

Ala?Pro?Arg?Thr?Pro?Gly?Gly?Arg?Arg

1???????????????5

<210>164

<211>8

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>164

Val?Glu?Arg?Tyr?Ala?Phe?Gly?Leu

1???????????????5

<210>165

<211>8

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>165

Leu?Pro?Val?Tyr?Asn?Phe?Gly?Leu

1???????????????5

<210>166

<211>11

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>166

Thr?Thr?Arg?Pro?Gln?Pro?Phe?Asn?Phe?Gly?Leu

1???????????????5???????????????????10

<210>167

<211>10

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>167

Glu?Asp?Val?Asp?His?Val?Phe?Leu?Arg?Phe

1???????????????5???????????????????10

<210>168

<211>7

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>168

Gly?Asn?Ser?Phe?Leu?Arg?Phe

1???????????????5

<210>169

<211>10

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>169

Ala?Pro?Thr?Ser?Ser?Phe?Ile?Gly?Met?Arg

1???????????????5???????????????????10

<210>170

<211>9

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>170

Ala?Pro?Leu?Ala?Phe?Tyr?Gly?Met?Arg

1???????????????5

<210>171

<211>9

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>171

Ala?Pro?Leu?Ala?Phe?Tyr?Gly?Leu?Arg

1???????????????5

<210>172

<211>9

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>172

Ala?Pro?Thr?Gly?Phe?Thr?Gly?Met?Arg

1???????????????5

<210>173

<211>10

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>173

Ala?Pro?Val?Asn?Ser?Phe?Val?Gly?Met?Arg

1???????????????5???????????????????10

<210>174

<211>9

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>174

Ala?Pro?Asn?Gly?Phe?Leu?Gly?Met?Arg

1???????????????5

<210>175

<211>8

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>175

Asp?Pro?Ala?Phe?Asn?Ser?Trp?Gly

1???????????????5

<210>176

<211>8

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>176

Gly?Ser?Gly?Phe?Ser?Ser?Trp?Gly

1???????????????5

<210>177

<211>8

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<220>

<221>MOD_RES

<222>(1)..(1)

＜223〉1 residue is pGlu.

<400>177

Xaa?Ser?Ser?Phe?His?Ser?Trp?Gly

1???????????????5

<210>178

<211>8

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>178

Gly?Ala?Ser?Phe?Tyr?Ser?Trp?Gly

1???????????????5

<210>179

<211>7

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>179

Asn?Pro?Phe?His?Ser?Trp?Gly

1???????????????5

<210>180

<211>7

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>180

Pro?Ser?Phe?His?Ser?Trp?Ser

1???????????????5

<210>181

<211>15

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>181

Asn?Ser?Val?Val?Leu?Gly?Lys?Lys?Gln?Arg?Phe?His?Ser?Trp?Gly

1???????????????5???????????????????10??????????????????15

<210>182

<211>15

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<220>

<221>MOD_RES

<222>(1)..(1)

＜223〉Xaa is pGlu.

<400>182

Xaa?Val?Arg?Phe?Arg?Gln?Cys?Tyr?Phe?Asn?Pro?Ile?Ser?Cys?Phe

1???????????????5???????????????????10??????????????????15

<210>183

<211>7

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>183

Gln?Arg?Phe?His?Ser?Trp?Gly

1???????????????5

<210>184

<211>14

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<220>

<221>MOD_RES

<222>(1)..(1)

＜223〉Xaa is pGlu

<221>DISULFID

<222>(7)..(14)

<400>184

Xaa?Val?Arg?Phe?Gln?Cys?Tyr?Phe?Asn?Pro?Ile?Ser?Cys?Phe

1???????????????5???????????????????10

<210>185

<211>15

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<220>

<221>MOD_RES

<222>(1)..(1)

＜223〉Xaa is pGlu.

<220>

<221>DISULFID

<222>(7)..(14)

<400>185

Xaa?Val?Arg?Tyr?Arg?Gln?Cys?Tyr?Phe?Asn?Pro?Ile?Ser?Cys?Phe

1???????????????5???????????????????10??????????????????15

<210>186

<211>11

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>186

Ala?Gln?Arg?Ser?Pro?Ser?Leu?Arg?Leu?Arg?Phe

1???????????????5???????????????????10

<210>187

<211>11

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>187

Pro?Ile?Arg?Ser?Pro?Ser?Leu?Arg?Leu?Arg?Phe

1???????????????5???????????????????10

Claims (48)

1. a method of identifying the correctives of combination between DmGPCR1 and the DmGPCR1 binding partners and/or function is characterized in that, comprises step:

(a) the DmGPCR1 binding partners is contacted under correctives compound existence of inferring or non-existent situation with the composition that contains DmGPCR1;

(b) combination between detection DmGPCR1 binding partners and the DmGPCR1; With

(c) determine in the presence of described supposition correctives compound, and compare under the non-existent situation of correctives compound of described supposition, in conjunction with or function be to increase or reduce,

The sequence of wherein said DmGPCR1 binding partners has at least 70% sequence homogeny with the sequence that is selected from SEQ ID NO:186 and SEQ IDNO:187.

2. the method for claim 1 is characterized in that, the sequence of described DmGPCR1 binding partners has at least 80% sequence homogeny with the sequence that is selected from SEQ ID NO:186 and SEQ ID NO:187.

3. the method for claim 1 is characterized in that, the sequence of described DmGPCR1 binding partners has at least 95% sequence homogeny with the sequence that is selected from SEQ ID NO:186 and SEQ ID NO:187.

4. the method for claim 1 is characterized in that, described DmGPCR1 binding partners has the sequence that is selected from SEQ ID NO:186 and SEQ ID NO:187.

5. method of controlling insect colony, it is characterized in that, comprise binding partners or the correctives of insect being used DmGPCR1 polynucleotide or polypeptide, to change expression or the activity of DmGPCR1, the sequence of wherein said binding partners has at least 70% sequence homogeny with the sequence that is selected from SEQ ID NO:186 and SEQ ID NO:187.

6. method as claimed in claim 5 is characterized in that, the sequence of described binding partners has at least 80% sequence homogeny with the sequence that is selected from SEQ IDNO:186 and SEQ ID NO:187.

7. method as claimed in claim 5 is characterized in that, the sequence of described binding partners has at least 95% sequence homogeny with the sequence that is selected from SEQ IDNO:186 and SEQ ID NO:187.

8. method as claimed in claim 5 is characterized in that, described binding partners has the sequence that is selected from SEQ IDNO:186 and SEQ ID NO:187.

9. method as claimed in claim 5 is characterized in that, described insect is selected from fly, fruit bat, tick, flea, louse, flat lice and cockroach.

10. treat or prevent in individuality because the disease that epizoa causes or the method for the patient's condition for one kind, it is characterized in that, comprise the DmGPCR1 binding partners to described individual administering therapeutic effective dose, wherein said DmGPCR1 binding partners has and has at least 70% sequence homogeny with the sequence that is selected from SEQ ID NO:186 and SEQ ID NO:187.

11. method as claimed in claim 10 is characterized in that, the sequence of described DmGPCR1 binding partners has at least 80% sequence homogeny with the sequence that is selected from SEQ ID NO:186 and SEQ ID NO:187.

12. method as claimed in claim 10 is characterized in that, the sequence of described DmGPCR1 binding partners has at least 95% sequence homogeny with the sequence that is selected from SEQ ID NO:186 and SEQ ID NO:187.

13. method as claimed in claim 10 is characterized in that, described DmGPCR1 binding partners has the sequence that is selected from SEQ ID NO:186 and SEQ ID NO:187.

14. method as claimed in claim 10 is characterized in that, described individuality is the people.

15.16. one kind makes DmGPCR and the method that the DmGPCR binding partners combines, and it is characterized in that, comprises step:

The composition that contains DmGPCR is contacted with the DmGPCR binding partners; With

Described DmGPCR binding partners is combined with described DmGPCR.

16.17. method as claimed in claim 15 is characterized in that, described DmGPCR is DmGPCR5 (SEQ ID NO:9).

17.18. method as claimed in claim 16 is characterized in that, described DmGPCR binding partners is fruit bat tachykinin (DTK).

18. method as claimed in claim 17, it is characterized in that, the sequence of described fruit bat tachykinin be selected from DTK-1 (SEQ ID NO:169), Met8-DTK-2 (SEQ ID NO:170), DTK-2 (SEQ ID NO:171), DTK-3 (SEQ ID NO:172), DTK-4 (SEQ ID NO:173), and the sequence of DTK-5 (SEQ ID NO:174) has at least 80% sequence homogeny.

19. method as claimed in claim 15 is characterized in that, described DmGPCR is DmGPCR7 (SEQID NO:17).

20. method as claimed in claim 19 is characterized in that, described DmGPCR binding partners is leucokinin (LK).

21. method as claimed in claim 20, it is characterized in that, the sequence of described fruit bat tachykinin be selected from LK-I (SEQ ID NO:175), LK-V (SEQ ID NO:176), LK-VI (SEQ ID NO:177), and LK-VIII (SEQ ID NO:178), culex kassinin kinin (SEQ ID NO:179), snail kassinin kinin (SEQ ID NO:180), DLK-1 (SEQ ID NO:181), DLK-2 (SEQ ID NO:182), DLK-2a (SEQ ID NO:183) has at least 80% sequence homogeny.

22. method as claimed in claim 15 is characterized in that, described DmGPCR is DmGPCR8 (SEQID NO:19).

23. method as claimed in claim 22 is characterized in that, described DmGPCR binding partners is an allatostatin.

24. method as claimed in claim 23 is characterized in that, the sequence of described allatostatin be selected from AST-C (SEQ ID NO:184 or DST-C SEQ ID NO:185) and have at least 80% sequence homogeny.

25. a method of identifying the correctives of combination between DmGPCR and the DmGPCR binding partners and/or function is characterized in that, comprises step:

The DmGPCR binding partners is contacted under correctives compound existence of inferring or non-existent situation with the composition that contains DmGPCR;

Detect the combination between DmGPCR binding partners and the DmGPCR;

Determine in the presence of described supposition correctives compound, and compare under the non-existent situation of correctives compound of described supposition, in conjunction with being to increase or reduce,

Determine that in the presence of described supposition correctives compound and compare under the non-existent situation of correctives compound of described supposition, function is to strengthen or weaken,

Wherein said DmGPCR is DmGPCR5 (SEQ ID NO:9).

26. method as claimed in claim 25 is characterized in that, described DmGPCR binding partners is the fruit bat tachykinin.

27. method as claimed in claim 26, it is characterized in that, the sequence of described fruit bat tachykinin be selected from DTK-1 (SEQ ID NO:169), Met8-DTK-2 (SEQ ID NO:170), DTK-2 (SEQ ID NO:171), DTK-3 (SEQ ID NO:172), DTK-4 (SEQ ID NO:173), and the sequence of DTK-5 (SEQ ID NO:174) has at least 80% sequence homogeny.

28. a method of identifying the correctives of combination between DmGPCR and the DmGPCR binding partners and/or function is characterized in that, comprises step:

The DmGPCR binding partners is contacted under correctives compound existence of inferring or non-existent situation with the composition that contains DmGPCR;

Detect the combination between DmGPCR binding partners and the DmGPCR; With

Determine in the presence of described supposition correctives compound, and compare under the non-existent situation of correctives compound of described supposition, in conjunction with being to increase or reduce,

Determine that in the presence of described supposition correctives compound and compare under the non-existent situation of correctives compound of described supposition, function is to strengthen or weaken,

Wherein said DmGPCR is DmGPCR7 (SEQ ID NO:17).

29. method as claimed in claim 28 is characterized in that, described DmGPCR binding partners is a leucokinin.

30. method as claimed in claim 29, it is characterized in that, the sequence of described leucocyte tachykinin be selected from LK-I (SEQ ID NO:175), LK-V (SEQ ID NO:176), LK-VI (SEQ ID NO:177), and LK-VIII (SEQ ID NO:178), culex kassinin kinin (SEQ ID NO:179), snail kassinin kinin (SEQ ID NO:180), DLK-1 (SEQ ID NO:181), DLK-2 (SEQ ID NO:182), DLK-2a (SEQ ID NO:183) has at least 80% sequence homogeny.

31. a method of identifying the correctives of combination between DmGPCR and the DmGPCR binding partners and/or function is characterized in that, comprises step:

The DmGPCR binding partners is contacted under correctives compound existence of inferring or non-existent situation with the composition that contains DmGPCR;

Detect the combination between DmGPCR binding partners and the DmGPCR; With

Determine in the presence of described supposition correctives compound, and compare under the non-existent situation of correctives compound of described supposition, in conjunction with being to increase or reduce,

Determine that in the presence of described supposition correctives compound and compare under the non-existent situation of correctives compound of described supposition, function is to strengthen or weaken,

Wherein said DmGPCR is DmGPCR8 (SEQ ID NO:19).

32. method as claimed in claim 31 is characterized in that, described DmGPCR binding partners is an allatostatin.

33. method as claimed in claim 32 is characterized in that, the sequence of described allatostatin has at least 80% sequence homogeny with the sequence that is selected from AST-C (SEQ ID NO:184 or DST-C SEQ ID NO:185).

34. a method of controlling insect colony is characterized in that, comprises binding partners or the correctives of insect being used DmGPCR polynucleotide or polypeptide, to change expression or the activity of DmGPCR

35. method as claimed in claim 34 is characterized in that, described insect is selected from fly, fruit bat, tick, flea, louse, flat lice and cockroach.

36. method as claimed in claim 34 is characterized in that, described DmGPCR binding partners is the fruit bat tachykinin.

37. method as claimed in claim 36, it is characterized in that, the sequence of described fruit bat tachykinin be selected from DTK-1 (SEQ ID NO:169), Met8-DTK-2 (SEQ ID NO:170), DTK-2 (SEQ ID NO:171), DTK-3 (SEQ ID NO:172), DTK-4 (SEQ ID NO:173), and the sequence of DTK-5 (SEQ ID NO:174) has at least 80% sequence homogeny.

38. method as claimed in claim 34 is characterized in that, described DmGPCR binding partners is a leucokinin.

39. method as claimed in claim 38, it is characterized in that, the sequence of described leucocyte tachykinin be selected from LK-I (SEQ ID NO:175), LK-V (SEQ ID NO:176), LK-VI (SEQ ID NO:177), and LK-VIII (SEQ ID NO:178), culex kassinin kinin (SEQ ID NO:179), snail kassinin kinin (SEQ ID NO:180), DLK-1 (SEQ ID NO:181), DLK-2 (SEQ ID NO:182), DLK-2a (SEQ ID NO:183) has at least 80% sequence homogeny.

40. method as claimed in claim 34 is characterized in that, described DmGPCR binding partners is an allatostatin.

41. method as claimed in claim 40 is characterized in that, the sequence of described allatostatin be selected from AST-C (SEQ ID NO:184 or DST-C SEQ ID NO:185) and have 80% sequence homogeny.

42. method as claimed in claim 34 is characterized in that, described DmGPCR correctives is anti--DmGPCR antibody, DmGPCR antisense polynucleotides or the non-peptide mimics of small-molecular weight.

43. method as claimed in claim 42 is characterized in that, the non-peptide mimics of described small-molecular weight is activator or antagonist.

44. treatment or prevention in individuality since the disease that causes of epizoa or the method for the patient's condition it is characterized in that, comprise DmGPCR binding partners to described individual administering therapeutic effective dose.

45. method as claimed in claim 44 is characterized in that, described individuality is companion animals, livestock, horse or people.

46. method as claimed in claim 44 is characterized in that, described binding partners is fruit bat tachykinin, leucokinin, allatostatin or antibody.

47. method as claimed in claim 44 is characterized in that, described binding partners is an antibody.

48. method as claimed in claim 47 is characterized in that, described antibody is chimeric antibody, CDR-grafted antibody, people's antibody or humanized antibody.