CN101040186A - Use of the receptor GPR86 - Google Patents

Abstract

We describe a method of identifying a molecule suitable for the treatment, prophylaxis or alleviation of a GPR86 associated disease, in particular inflammatory disease or pain, the method comprising determining whether a candidate molecule is an agonist or antagonist of GPR86 polypeptide, in which the GPR86 polypeptide comprises the amino acid sequence shown in SEQ ID NO: 3 or SEQ ID NO: 5 or SEQ ID NO: 7, a fragment thereof or a sequence which is at least 90% identical thereto.

Description

The purposes of the receptor GPR 86

The field

The present invention relates to nucleic acid and by the new evaluation function of their encoded polypeptides, and their production and purposes.In particular, nucleic acid and polypeptide relate to g protein coupled receptor (GPCR), hereinafter referred to as the member of the purinoceptor family of " GPR86 " and GPCR.The invention still further relates to the effect that suppresses or activate this nucleic acid and polypeptide.

Background

Clearly determined many medically important biological processes be by participate in relating to G albumen and/or second messenger for example the signal transduction pathway of cAMP protein mediation (Lefkowitz, Nature, 1991,351:353-354).These protein are called protein or " the PPG albumen " that participates in G albumen approach.Some examples of these protein comprise gpc receptor, such as those acceptors (Kobilka, B.K.et al., Proc.Natl.Acad.Sci.USA, 1987, the 84:46-50 of adrenergic medicament and dopamine; Kobilka, B.K.et al., Science, 1987,238:650-656; Bunzow, J.R.et al., Nature, 1988,336:783-787), G albumen itself, effector molecules protein for example phospholipase C, adenyl cyclase and phosphodiesterase, and drive thing (actuator) protein for example protein kinase A and protein kinase C (Simon, M.I.et al., Science, 1991,252:802-8).

For example, in a kind of signal transduction of form, the effect of hormone combination is the adenyl cyclase of activating cell inside.Hormone depends on the existence of nucleotide GTP to the activation of enzyme.GTP also influences the hormone combination.G albumen is connected to hormone receptor on the adenyl cyclase.When G albumen is subjected to the hormone receptor activation according to the show, the GDP of combination is exchanged into GTP.The form of carrying GTP then is just in conjunction with the adenyl cyclase that activates.The catalysis GTP of G albumen own is hydrolyzed into GDP, makes G albumen turn back to the inactive form on basis.Therefore, G albumen is as signal is relayed the intermediate of effector and the timer of control signal duration from acceptor and serve a dual purpose.

The membrane protein gene superfamily of g protein coupled receptor (GPCR) has identified has seven membrane-spanning domains of inferring.Think that the representative of these domains strides film α spiral by what born of the same parents' outer shroud or kytoplasm ring connected.G protein coupled receptor comprises far-ranging biologic activity acceptor, such as hormone, virus, growth factor and neuroceptor.

G protein coupled receptor (having another name called the 7TM acceptor) has been accredited as and has comprised about 20-30 amino acid whose these seven conservative hydrophobic fragments, connects the hydrophilic loop of at least eight dispersions.The G protein family of coupled receptor comprises the dopamine receptor in conjunction with the neuroleptic that is used for the treatment of spirit and neurological disorder.Other example of this family member is including, but not limited to calcitonin, adrenaline, Endothelin, adenosine, muscarine (muscarinic), thrombocytin, histamine, fibrin ferment, kassinin kinin, follicle-stimulating hormone (FSH), opsin, endothelial differentiation gene-1, rhodopsin, smell (odorant) and cytomegalovirus acceptor.

Most of g protein coupled receptors have single conservative cysteine residues at preceding two born of the same parents' outer shrouds in each, think their formed disulfide bond stabilization function protein structures.Stride film district called after TM1, TM2, TM3, TM4, TM5, TM6 and TM7 for 7.TM3 connects with signal transduction.

The phosphorylation of cysteine residues and fatization (lipidation) (palmitylization (palmitylation) or farnesylation (farnesylation)) can influence the signal transduction of some g protein coupled receptor.Most of g protein coupled receptors comprise potential phosphorylation site in the 3rd kytoplasm ring and/or carboxyl terminal.For several g protein coupled receptors, such as receptor,, the phosphorylation of protein kinase A and/or special receptor kinase mediation receptor desensitization.For some acceptor, think that the ligand-binding site point of g protein coupled receptor comprises the hydrophilic groove that is formed by several g protein coupled receptor membrane-spanning domains, this groove is surrounded by the hydrophobic residue of g protein coupled receptor.Think the hydrophilic surface of each g protein coupled receptor transbilayer helix all towards inside, and form the ligand-binding site point of polarity.TM3 is because have the ligand-binding site point, such as the TM3 asparagicacid residue, and relevant with several g protein coupled receptors.TM5 serine, TM6 asparagine and TM6 or TM7 phenylalanine or tyrosine also with part in conjunction with relevant.

G protein coupled receptor can by the various endocellular enzymes of heterotrimeric G protein coupling in born of the same parents, ion channel and transport protein (referring to Johnson et al., Endoc.Rev., 1989,10:317-331).Different G protein alpha subunits preferentially stimulates the specific effect device to regulate various biological functions in cell.The phosphorylation of having identified g protein coupled receptor tenuigenin residue is the important mechanisms of regulating some g protein coupled receptor of G albumen coupling.G protein coupled receptor has been found at many positions in mammalian hosts.In in the past 15 years, nearly 350 kinds of targets 7 are striden the therapeutic agent of film (7TM) acceptor and have successfully been introduced market.

Therefore, g protein coupled receptor has the history of conduct treatment target definite, the process proof.Obviously, need to identify and characterize the more polyceptor that can in prevention, improvement or arbor press dysfunction or disease, work.

General introduction

According to a first aspect of the present invention, we provide evaluation to be suitable for treatment, to prevent or to alleviate the GPR86 relevant disease, the method of the molecule of inflammatory disease or pain particularly, this method comprises determining whether candidate molecules is the activator or the antagonist of GPR86 polypeptide, wherein said GPR86 polypeptide comprise amino acid sequence shown in SEQ ID NO:3, SEQ ID NO:5 or the SEQ ID NO:7, its fragment or with its at least 90% identical sequence.

Preferably, described GPR86 polypeptide is by nucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:2 or the SEQID NO:4 or with its at least 90% identical sequential coding.

Preferably, this method comprises that candidate molecules is exposed to the GPR86 polypeptide determines that also whether candidate molecules is in conjunction with the GPR86 polypeptide.

Preferably, the cells contacting candidate compound of the G Protein G i coupling by making contained GPR86 acceptor and GPR86 preference and the level of determining GPCR sensitivity label thing such as cyclic adenosine monophosphate (cAMP) in the described cell whether since described contact to reduce identify activator.

Preferably, the cells contacting candidate compound of the G Protein G i coupling by making contained GPR86 acceptor and GPR86 preference and the level of determining GPCR sensitivity label thing such as cyclic adenosine monophosphate (cAMP) in the described cell whether since described contact to improve identify antagonist.

Preferably, by making contained GPR86 acceptor and the pungency G albumen that mixes such as G _{α 16}The cells contacting candidate compound of coupling also determines whether the level of GPCR sensitivity label thing such as cyclic adenosine monophosphate (cAMP) in the described cell identifies activator owing to described contact improves.

Preferably, by making contained GPR86 acceptor and the pungency G albumen that mixes such as G _{α 16}The cells contacting candidate compound of coupling also determines whether the level of GPCR sensitivity label thing such as cyclic adenosine monophosphate (cAMP) in the described cell identifies antagonist owing to described contact reduces.

Preferably, this method comprises the transgenic nonhuman animal that (a) provides the function of wild type animal or endogenous GPR86 gene to be destroyed; (b) wild type or transgenic nonhuman animal are exposed to candidate molecules; And whether the biological parameter of (c) determining animal is owing to described contact changes.

Preferably, described biological parameter be selected to the reaction that stimulates, to the reaction of heat, to light reaction, immune response, inflammatory reaction, to the reaction of pain, preferably to the reaction of pain.

Preferably, this method comprises the cell that (a) provides the function of wild-type cell or endogenous GPR86 gene to be destroyed, preferably isolated cells from the transgenic nonhuman animal that the function of endogenous GPR86 gene is destroyed; (b) with cellular exposure in candidate molecules; And whether the biologic activity of (c) determining the GPR86 polypeptide is owing to described contact changes.

According to a second aspect of the present invention, the transgenic nonhuman animal that provides the function of wild type animal or endogenous GPR86 gene to be destroyed is used for the treatment of, prevents in evaluation or alleviates purposes in the method for the GPR86 polypeptide activator of GPR86 relevant disease, particularly inflammatory disease or pain or antagonist.

The transgenic nonhuman animal that the function of endogenous GPR86 gene is destroyed or its isolated cells or tissue are as the purposes of GPR86 relevant disease, particularly inflammatory disease or pain model.

Preferably, described transgenic nonhuman animal comprises the GPR86 gene that function is destroyed, and preferably comprises deletion in GPR86 gene or its part.

Preferably, compare with the wild type animal, transgenic nonhuman animal demonstrates variation in following each or multinomial phenotype: to the reaction that stimulates, to the reaction of heat, to light reaction, immune response, inflammatory reaction, to the reaction of pain, preferably to the reaction of pain.

Preferably, compare with the wild type animal, transgenic nonhuman animal demonstrates following at least one: (a) neurological susceptibility to pain changes, and promptly the susceptibility to pain improves or reduces, (b) neurological susceptibility to inflammatory pain changes, and promptly the neurological susceptibility to inflammatory pain improves or reduces.

Preferably, described transgenic nonhuman animal is a rodent, preferred mouse.

According to a third aspect of the present invention, we provide the activator of identifying the GPR86 polypeptide or the method for antagonist, and this method comprises the variation of using candidate compound and measure biological parameter to described wild type or transgenic nonhuman animal.

As a fourth aspect of the present invention, provide to comprise amino acid sequence shown in SEQ ID NO:3, SEQ ID NO:5 or the SEQ ID NO:7, its fragment or identify and be used for the treatment of, prevented its activator of GPR86 relevant disease, particularly inflammatory disease or pain or the purposes of antagonist with the GPR86 polypeptide of its at least 90% identical sequence.

According to a fifth aspect of the present invention, we provide to comprise nucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:2 or the SEQ ID NO:4, its fragment or identify with the GPR86 polynucleotide of its at least 90% identical sequence and have been used for the treatment of, have prevented its activator of GPR86 relevant disease, particularly inflammatory disease or pain or the purposes of antagonist.

Preferably, described pain is selected from acute pain, chronic pain, dermatalgia, somatalgia, splanchnodynia, referred pain comprises myocardial ischemia, unreal painful and neuropathic pain (neuralgia), by damage, the pain that disease causes, headache, antimigraine, cancer pain, the pain that causes by neurological disorder such as op parkinson's (Parkinson) disease, by backbone and operation on peripheral nerve, brain tumor, traumatic brain injury (TBI), trauma of spinal cord, chronic pain syndrome, the pain that chronic fatigue syndrome causes, neuralgia is such as trigeminal neuralgia, glossopharyngeal neuralgia, postherpetic neuralgia and causalgia, by lupus, sarcoidosis, archnoiditis, arthritis, the pain that rheumatic disease causes, periodicity pain, backache, back pain, arthralgia, stomachache, pectoralgia, pain of childbirth, muscle skeleton and disease of skin, head trauma and fibromyalgia.

Preferably, described inflammatory disease is selected from the inflammatory disorder, is preferably selected from inflammatory disease (rheumatoid arthritis for example, multiple sclerosis, Ge-Ba Er Shi (Guillain-Barre) syndrome, Crow engler (Crohn) disease, ulcerative colitis, psoriasis, graft versus host disease(GVH disease), systemic lupus, lupus erythematosus (erythematosus) or insulin-dependent diabetes), autoimmune disease (TSS for example, osteoarthritis, diabetes or inflammatory bowel disease), acute pain, chronic pain, neuropathic pain, contact dermatitis, atherosclerotic, glomerulonephritis, reperfusion injury, bone-resorbing disease, asthma, apoplexy, miocardial infarction, thermal burn, adult respiratory distress syndrome (ARDS) (ARDS), the multiple organ injury of wound secondary, skin disease with the acute inflammation composition, acute purulent meningitis, gangrenosum acne intestines colitis (necrotising entrerocolitis), with haemodialysis, infectious shock (septic shock), leucocyte removal art (leukophcrisis), the syndrome that the granulocyte blood transfusion is relevant, suck the acute or chronic inflammation of lung that causes by smog, mullerianosis, Bei Qieteshi (Behcet) disease, uveitis, ankylosing spondylitis, pancreatitis, cancer, lime (Lyme) disease, ISR after skin is worn chamber coronary angioplasty (percutaneous transluminal coronary angioplasty), Alzheimers (Alzheimer) disease, traumatic arthritis, pyemia, chronic obstructive pulmonary disease, congestive heart failure, osteoporosis, cachexia, op parkinson's (Parkinson) disease, periodontosis, gout, allergic disease, the age related macular degeneration, infect and cystic fibrosis.

Aspect the 6th, the invention provides GPR86 activator or the antagonist of identifying by listed method or purposes.

In a seventh aspect of the present invention, provide this quasi-molecule to be used for the treatment of, to prevent or alleviate the purposes of inflammatory disease or pain.

According to a eighth aspect of the present invention, we provide the diagnostic kit of inflammatory disease or pain or its neurological susceptibility, comprise following each or multinomial: GPR86 polypeptide or its part; Antibody at the GPR86 polypeptide; Maybe can the encode nucleic acid of above-mentioned substance.

According to a ninth aspect of the present invention, we provide treatment to suffer from the method for the individuality of inflammatory disease or pain, and this method comprises activity or the content that improves or reduce GPR86 polypeptide in the individuality.

Preferably, this method comprises to individuality and uses GPR86 polypeptide, GPR86 polypeptide activator or GPR86 antagonist.

According to a tenth aspect of the present invention, the method for diagnosis inflammatory disease or pain is provided, the method comprising the steps of: (a) detect and suffer from or suspect GPR86 polypeptide expression level or pattern in the animal that suffers from this type of disease; And (b) expression or pattern and intact animal are compared.

The accompanying drawing summary

Fig. 1 has shown the result of the HMM structure prediction software analysis people GPR86 polypeptide (SEQ ID NO:3) that utilizes pfam (http://www.sanger.ac.uk/Software/Pfam/search.shtml).

Fig. 2 has shown the synoptic diagram that knocks out plasmid.

Fig. 3 has shown the express spectra of the people GPR86 that generates by reverse-transcription polymerase chain reaction (RT-PCR).

Fig. 4 is the data plot that obtains from the test of wagging the tail (Tail Flick Test), has shown GPR86 knock-out animal and wild type animal result relatively.

Fig. 5 is the data plot that obtains from formalin test (Formalin Test), has shown GPR86 knock-out animal (white) and wild type animal (black) result relatively with the wide increased percentage of pawl.

Fig. 6 be electronics Vonfrey test (Electronic Vonfrey Test) figure as a result (/-knock-out animal; + /+wild type contrast).

Fig. 7 has shown the RT-PCR analysis result that GPR86 expresses in many people's derived tissues.The 1st road: marrow; The 2nd road: thymus gland; The 3rd road: lymph node; The 4th road: Jurkat CD4+; The 5th road: Myla CD8+; The 6th road: Colo 720; The 7th road: THP1; The 8th road: Gegenbaur's cell; The 9th road: cartilage cell; The 10th road: negative control; The 11st road: positive control (brain).

Fig. 8 has shown the RT-PCR analysis result that GPR86 expresses in many mouse-derived tissues.The 1st road: spleen; The 2nd road: salivary gland; The 3rd road: spinal cord; The 4th road: muscle; The 5th road: tongue; The 6th road: ovary; The 7th road: pancreas; The 8th road: fat; The 9th road: testis; The 10th road: the heart; The 11st road: eye; The 12nd road: lung; The 13rd road: kidney; The 14th road: thymus gland; The 15th road: stomach+SI; The 16th road: brain; The 17th road: liver+Gb; The 18th road: blood; The 19th road: bladder; The 20th road: adrenal gland; The 21st road: C57BL6J genomic DNA; The 22nd road: 129SvEv genomic DNA.

Sequence table

SEQ ID NO:1 shows the cDNA sequence of people GPR86.SEQ ID NO:2 shows the open read frame that derives from SEQ ID NO:1.SEQ ID NO:3 shows the amino acid sequence of people GPR86.SEQ IDNO:4 shows the cDNA open read frame of mouse GPR86.SEQ ID NO:5 shows the amino acid sequence of mouse GPR86.SEQ ID NO:6 shows the variable cDNA sequence of people GPR86.SEQ ID NO:7 shows the variable amino acid sequence of people GPR86.SEQ ID NO:8-20 demonstration knocks out the plasmid primer sequence.SEQ ID NO:21 demonstration knocks out plasmid sequence.

The method that adopts

Unless otherwise stated, otherwise implement the present invention and will adopt those of ordinary skills' limit of power interior chemistry, molecular biology, microbiology, recombinant DNA and immunologic routine techniques.In the document these technology are explained.Referring to for example J.Sambrook, E.F.Fritsch, and T.Maniatis, 1989, Molecular Cloning:A Laboratory Manual, Second Edition, Books 1-3, Cold Spring Harbor Laboratory Press; Ausubel, F.M.et al. (1995 and periodicsupplements; Current Protocols in Molecular Biology, ch.9,13, and 16, JohnWiley ﹠amp; Sons, New York, N.Y.); B.Roe, J.Crabtree, and A.Kahn, 1996, DNAIsolation and Sequencing:Essential Techniques, John Wiley ﹠amp; Sons; J.M.Polakand James O ' D.McGee, 1990, In Situ Hybridization:Principles and Practice, Oxford University Press; M.J.Gait (Editor), 1984, Oligonucleotide Synthesis:APractical Approach, Irl Press; D.M.J.Lilley and J.E.Dahlberg, 1992, Methodsof Enzymology:DNA Structure Part A:Synthesis and Physical Analysis of DNA, Methods in Enzymology, Academic Press; Using Antibodies:A LaboratoryManual:Portable Protocol NO.I by Edward Harlow, David Lane, Ed Harlow (1999, Cold Spring Harbor Laboratory Press, ISBN 0-87969-544-7); Antibodies:A Laboratory Manual by Ed Harlow (Editor), David Lane (Editor) (1988, ColdSpring Harbor Laboratory Press, ISBN 0-87969-314-2), 1855, Lars-Inge Larsson " Immunocytochemistry:Theory and Practice ", CRC Press inc., Baca Raton, Florida, 1988, ISBN 0-8493-6078-1, John D.Pound (ed); " ImmunochemicalProtocols, vol 80 ", in the series: " Methods in Molecular Biology ", Humana Press, Totowa, New Jersey, 1998, ISBN 0-89603-493-3, Handbook of Drug Screening, edited by Ramakrishna Seethala, Prabhavathi B.Fernandes (2001, New York, NY, Marcel Dekker, ISBN 0-8247-0562-9); Lab Ref:A Handbook of Recipes, Reagents, and Other Reference Tools for Use at the Bench, Edited Jane Roskamsand Linda Rodgers, 2002, Cold Spring Harbor Laboratory, ISBN 0-87969-630-3; And The Merck Manual of Diagnosis and Therapy (17th Edition, Beers, M.H., andBerkow, R, Eds, ISBN:0911910107, John Wiley ﹠amp; Sons).These common textbooks all are collected herein by reference.

Describe in detail

GPR86

Described g protein coupled receptor (GPCR) on this part file population, particularly we are called the purinoceptor type g protein coupled receptor of GPR86, and homolog, variant or derivant.

We have determined that compare with the wild type animal, the transgenic animals that lack functional GPR86 demonstrate the neurological susceptibility of change to pain.Particularly, the GPR86 knock-out animal is more insensitive to pain.And described animal demonstrates the neurological susceptibility of change to inflammatory pain, and particularly the neurological susceptibility to inflammatory pain reduces.

Therefore, we disclose GPR86, its homolog, variant or derivant, and instrumentality is in the purposes for the treatment of, alleviate or diagnosing in the pain.Hereinafter with more detailed description this embodiment of the present invention and other embodiment.

The express spectra of GPR86

Go out GPR86 at people's derived tissues by polymerase chain reaction (PCR) augmentation detection to GPR86 cDNA, the expression abundance among marrow, thymus gland, lymph node, leucocyte, Gegenbaur's cell and the cartilage cell is different.At two people's derived cells derived from the T cell is also to have found it among Jurkat CD4+ and the Myla CD8+.Observing low-level expression derived from lymphocytic Colo720 with in derived from monocytic THP1 cell.(embodiment 4; Fig. 7).

And at mouse tissue, (embodiment 4, Fig. 8) also to detect the expression of GPR86 in spleen, salivary gland, spinal cord, tongue, fat, testis, the heart, eye, lung, kidney, thymus gland, stomach and small intestine, brain, liver and gall-bladder, blood, bladder and the adrenal gland.

Fig. 3 has shown the express spectra of GPR86.

Utilize the GPR86 cDNA seeker EST data source of SEQ ID NO:1 and 6 by BLASTN, finding homogeneity by being derived among the cDNA that derives in the popular feeling, placenta and colon and mouse skin, breast and hypothalamic storehouse.This shows that GPR86 normally or in the abnormal structure expresses at these.Therefore, GPR86 polypeptide, nucleic acid, probe, antibody, expression vector and part can be used for these and other tissue in cross the expressing of GPR86, the low expression and detection, diagnosis, treatment and other determination method of unconventionality expression diseases associated.

The GPR86 relevant disease

According to methods described herein and composition, GPR86 GPCR can be used for the treatment and the diagnosis of numerous disease.For simplicity, these diseases are called " GPR86 relevant disease ".

Therefore, the GPR86 deficient animals can be used as the model of GPR86 relevant disease.GPR86, its fragment, homolog, variant and derivant, and instrumentality can be used for diagnosis or treatment GPR86 relevant disease particularly including activator and antagonist.Particularly, GPR86 can be used for screening the molecule that can influence its function, and this molecule can be used for treating the GPR86 relevant disease.

We have proved that here people GPR86 is positioned human chromosomal 3q24.Therefore, in a specific embodiment, GPR86 can be used for treatment or diagnosis is positioned this locus, chromosome band, district, arm or this chromosomal disease.

Determined to comprise following (being the position in the bracket): acute myeloid leukaemia (3q24), Theo Hermans Ji-Pu De clarke (Hermansky-Pudlak) syndrome (3q24), platelet ADP receptor defective (3q24-q25) and Dan Di-Wo Ke (Dandy Walker) syndrome (3q24) with the known disease of homologous genes seat, chromosome band, district, arm or the chromosome linkage of the chromosome position (being human chromosomal 3q24) of GPR86.

Therefore, according to an embodiment preferred, by any means of describing in the presents, GPR86 and instrumentality thereof (such as activator and antagonist) can be used for diagnosis or treatment dopamine relevant disease, such as Parkinson's disease, heart disease such as supraventricular or ventricular arrhythmia, low blood pressure, feel sick, tourette many (Tourette) syndrome, stress and pain.

As proving among the embodiment, the knock-out mice of GPR86 defective shows multiple phenotype.

Particularly, embodiment 5 and Fig. 4 have proved that in the test of wagging the tail, the knock-out animal that lacks functional GPR86 is than the wild type animal, and stimulation and pain are more insensitive to external world.Similarly, embodiment 7 and Fig. 6 have shown that in the test of Von Frey filament, the knock-out animal that lacks functional GPR86 is than the wild type animal, and stimulation and pain are more insensitive to external world.

Therefore, according to a preferred embodiment of the present invention, by any means of describing in the presents, GPR86 and instrumentality thereof (such as activator and preferred antagonist) can be used for diagnosis or treatment pain and cancer.Particularly, pain comprises neuropathic pain, postherpetic neuralgia, the diabetes nerve pain, trigeminal neuralgia, alcohol (ethanol) related neural disease, neuralgia leprous, vasculitis neuralgia, uremia neuralgia, Ge-Ba Er Shi (Guillain Barre) syndrome, multiple sclerosis, acute immune neuropathy, thoracic outlet syndrome, carpal tunnel syndrome, tarsal tunnel syndrome, meralgia paraesthetica, the recombination region pain syndrome, temporal-mandible joint syndrome (temperomandibular joint syndrome), atypical facial pain, back pain, lumbar spine is narrow, interverbebral disc (disc) disease.Cervicodynia, the scorching MN of cervical spine, cervical spondylopathy, cervical intervertebral disk disease, cervical spinal prosopodynia (cervical myelofacial pain).Inflammatory pain, osteoarthritis, rheumatoid arthritis, inflammatory bowel disease.Cancer pain, cancer in particular comprise breast cancer, prostate cancer, colon cancer, lung cancer, oophoroma and osteocarcinoma.Comprise headache, antimigraine, tension headache, cluster headache, chronic paroxysmal hemicrania, and splanchnodynia, dysmenorrhoea, non-pepsin indigestion, non-cardiac chest pain, IBS, rectum phantom pain.Thermal hyperalgesia and postoperative pain.

And we have proved that in an embodiment GPR86 expresses (embodiment 3 and 4) in derived from the cell of immune response cell.As embodiment 6 and shown in Figure 5, the transgenic animals that lack functional GPR86 demonstrate the inflammation tendency and reduce.This has proved that GPR86 is relevant with inflammatory reaction, and described inflammatory reaction comprises the inflammatory that those relate to pain and the inflammatory reaction of nerve aspect.

According to another aspect, by any means of describing in the presents, GPR86 and instrumentality thereof (such as activator and antagonist) can be used for diagnosis or treatment inflammatory disease (rheumatoid arthritis for example, multiple sclerosis, guillain-Barre syndrome, Crohn's disease, ulcerative colitis, psoriasis, graft versus host disease(GVH disease), systemic lupus, lupus erythematosus or insulin-dependent diabetes), autoimmune disease (TSS for example, osteoarthritis, diabetes or inflammatory bowel disease), acute pain, chronic pain, neuropathic pain, contact dermatitis, atherosclerotic, glomerulonephritis, reperfusion injury, bone-resorbing disease, asthma, apoplexy, miocardial infarction, thermal burn, adult respiratory distress syndrome (ARDS) (ARDS), the multiple organ injury of wound secondary, skin disease with the acute inflammation composition, acute purulent meningitis, gangrenosum acne intestines colitis, with haemodialysis, infectious shock, the leucocyte removal art, the syndrome that the granulocyte blood transfusion is relevant, suck the acute or chronic inflammation of lung that causes by smog, mullerianosis, Behcet, uveitis, ankylosing spondylitis, pancreatitis, cancer, Lyme disease, ISR after skin is worn the chamber coronary angioplasty, Alzheimer's, traumatic arthritis, pyemia, chronic obstructive pulmonary disease, congestive heart failure, osteoporosis, cachexia, Parkinson's disease, periodontosis, gout, allergic disease, the age related macular degeneration, infect and cystic fibrosis.

For convenience, the disease with available GPR86 treatment and/or diagnosis is called " GPR86 relevant disease ".In particularly preferred embodiments, the GPR86 relevant disease comprises that those comprise the disease (referring to above-mentioned paragraph) of pain as symptom.

As mentioned above, use any method and composition described herein, GPR86 and instrumentality thereof (such as activator and antagonist) can be used for diagnosing and/or treat the concrete disease of any of these.

Particularly, the transgenic animals that we have specifically looked forward to nucleic acid, the carrier, the polypeptide that comprise GPR86 nucleic acid comprises its homolog, variant or derivant, Pharmaceutical composition, host cell and comprise GPR86 nucleic acid and/or polypeptide are used for the treatment of or diagnose the above purposes of listed disease specific.And we have looked forward to can or combine and can change the compound of endogenous cAMP level with the GPR86 interaction, at antibody and the preparation of GPR86 or the method for identifying these compounds and antibody in diagnosis or treat purposes in the above-mentioned disease specific.Particularly, we comprise that any of these compound, composition, molecule etc. are used for the treatment of or prevent purposes in the vaccine of disease specific in production.We also disclose the diagnostic kit that is used for detecting at individuality disease specific.

The linkage mapping method of these or other disease specific of available GPR86 treatment or diagnosis is known in this area, and also is described in other place of presents.

Pain

GPR86 that describes in the presents and variant thereof, and their sequence, its antibody etc. of encoding can be used for diagnosis and/or treat many and pain diseases associated.

Acute pain

Acute pain is defined as short term pain or has the pain of the cause of disease that is easy to recognize.Acute pain is that health is to the disorganization of existence or the warning of disease.It usually is quick and violent, follow-up pain.Acute pain concentrates on a zone earlier, slightly launches then.

Chronic pain

Chronic pain medically be defined as continued 6 months or the longer time pain.This pain lasting or intermittence is usually permanent than its purpose, so it can not help the pre-antisitic defect of health.It is usually than more refractory treatment of acute pain.Special and professional care treats normally that to have become chronic any pain necessary.Behind long-term use opioid, drug resistance, drug dependence and even psychological habituation may take place.Though drug resistance and drug dependence are common in the opioid user, psychological habituation is rare.

Can the experience of physiology pain be divided into four classes according to source and relevant nociceptor (pain detect neural).

Dermatalgia

Dermatalgia is caused by skin or superficial tissues damage.The skin damage receptor just in time stops under skin, and owing to the nerve endings of high concentration produces the local pain that the duration is short, the division is clearly demarcated.The damage example that produces dermatalgia comprises that paper scratches, slight (once) burns and tears.

Somatalgia

Somatalgia be derived from ligament, tendon, bone, blood vessel and even neural itself, and detect by the body nociceptor.These regional pain receptors are not enough and it is longer than the dermatalgia duration to produce, can not pinpoint dull pain; Example comprises sprain of ankle and bone fracture.

Splanchnodynia

The splanchnodynia that is derived from body organ internal organ nociceptor is positioned in body organ and the inner body cavity.These regional nociceptors even not enough more and produce usually than somatalgia more pain and longer pain of duration.The extremely difficult location of splanchnodynia, and several damages of viscera tissue are demonstrated " involving property " pain, that is that pain is positioned and the damage location sensation in irrelevant zone fully.Myocardial ischemia (the blood flow loss of part cardiac muscular tissue) may be the example of well-known telalgia; Sensation can occur in upper breast with restriction touch form, perhaps occurs in left shoulder, arm or even hand with the pain form.

The pain of other type

Phantom limb pain is from no longer including or the pain sensation-amputee and the almost generally experience of report of quadriplegic of the limbs of the physical signalling of not reentrying.The generation of neuropathic pain (neuralgia) can be the damage of nerve fiber itself or the result of disease.This can destroy sensory nerve and transmit the ability of correct information to thalamus, and therefore even without pain physiological reason obvious or record, brain still can be thought pain stimulation.

Trigeminal neuralgia refers to the pain that caused by injury of trigeminal nerve or destruction.Trigeminal neuralgia has 3 branch road: V1 to produce the sensation in forehead and eye district, and V2 produces the sensation of nose and face, and V3 produces the sensation in jaw (jaw) and chin (chin) district.The every side of face all has sensigenous trigeminal neuralgia.The trigeminal neuralgia of one side is extensible through cheek (cheek), mouth, nose and/or jaw flesh.Also may experience trigeminal neuralgia though young man or those suffer from the people of multiple sclerosis, trigeminal neuralgia is the old people of influence usually.

Prosopalgic cardinal symptom is the pain of forehead, cheek, chin or jaw profile.Cases with severe can relate to whole three zones or the left and right sides.Pain outbreak is serious, be spastic and the time short, be described as feeling similar when getting an electric shock.Daily routines are such as brushing teeth, talk, chew, drink water, shave or even kissing and can both trigger pain.The frequency of pain outbreak increases in time, becomes more disruptive and disables.

Glossopharyngeal neuralgia is to be the clinical entity of feature with the outburst of the pain of the sensation distributive province of cranial nerve,ninth.Except that pain position and pain stimulation, this pain outbreak is identical with trigeminal neuralgia.Typical case's pain is the repeated serial pain that the serious lancinating picture electricity of a flat-sided peach body region or back of tongue is stabbed.In addition, pain is radiation-curable to ear or be derived from ear.

The sensory stimuli of bringing out pain is to swallow, and during serious attack, the patient can sit quietly, and is anteflexion, and the saliva freedom is flowed out from mouth.Heartbeat stops, fainting (fainting) and epileptic attack connects with the glossopharyngeal neuralgia outbreak.The cause of disease of glossopharyngeal neuralgia is unknown in the most applications.But, some cases have been belonged to tumour, vertebral artery compressing and vascular malformation to cranial nerve,ninth.

Postherpetic neuralgia refers to infect the chronic ache that continues behind the herpes zoster virus.Herpes zoster is the recurrent infection of varicella-zoster (varicella) virus infections.Virus lays dormant weakens up to patient's immunity in neural.The acute pathology of herpes zoster causes the pain that can disappear usually.But in many patients, pain can continue-postherpetic neuralgia for a long time.

The symptom of herpes zoster comprises the continuous dark pain of tearing: pain is positioned at chest 65% and face 20%.When relating to face, virus demonstrates the preference (the face top that eyebrow is above) of ophthalmic division of trigeminal nerve.Pain is usually at 2-4 week spontaneous regression.But small number of patients will have constant pain.Pain is positioned at former fash district, but strokes gently that ill skin can aggravate the pain and to this zone alleviating pain of exerting pressure.The friction of clothes usually is very bitterly.This constant pain is called postherpetic neuralgia.The incidence of disease that herpes zoster involves postherpetic neuralgia under the facial situation is higher.

Cusalgia is the rare syndrome after the part peripheral nerve injury.Its feature is the triad that burn pain, autonomous dysfunction and nutrition change.Serious situation is called severe (major) cusalgia.Slightly (minor) cusalgia shows as the more not serious form similar to reflex sympathetic dystrophy (RSD).RSD has outstanding muscle and joint symptom, common osteoporosis on X ray.

Cusalgia causes by peripheral nerve injury, normally brachial plexus injury.Denervate causes supersensitivity, cause pain to increase, and norepinephrine discharges the result (sympathetic findings) that increase causes the sympathetic nerve aspect.Symptom comprises pain: normally burn sense, and also remarkable in hand or the foot.Most of people are damaging outbreak in 24 hours.The most normal median nerve, ulnar nerve and the sciatic nerve of relating to.Almost any sensory stimuli all worsens pain.Blood vessel changes: make blood increase (warm, pink) or make blood reduce (cold, assorted blue) by vessel retraction by vasodilation.Nutrition changes: and drying/flaky skin, ankyloses, finger tip be thin, carinate does not trim the nails, hair is long/and thick or trichomadesis, perspiration change.

Homogeneity and similarity with GPR86

GPR86 structurally has relation with other protein of g protein coupled receptor family, shown in the sequencing result of the cDNA product of the coding people GPR86 of amplification.The cDNA sequence of SEQ ID NO:1 comprises the open read frame (SEQ ID NO:2, nucleotide numbering 19-1084) of 354 amino acid whose polypeptide shown in the coding SEQ ID NO:3.Finder GPR86 is positioned human chromosomal 3q24.Polypeptide shown in the variable cDNA sequential coding SEQ ID NO:7 of SEQ ID NO:6.

P2Y12 platelet ADP receptor [mankind] homogeneity=154/316 (48%), positive=211/316 (66%).The g protein coupled receptor of KIAA0001 supposition; The g protein coupled receptor of UDP-glucose; Homogeneity=140/295 (47%), positive=193/295 (64%).

Platelet activation acceptor homolog [mankind]; Homogeneity=42/144 (29%), positive=78/144 (54%)

The HMM structure prediction software of use pfam (http://www.sanger.ac.uk/Software/Pfam/search.shtml) has confirmed that to the analysis of GPR86 polypeptide (SEQ ID NO:3) the GPR86 peptide is the GPCR (referring to Fig. 1) of 7TM-1 structure class.

Cloned the mouse homolog of people GPR86, its nucleotide sequence and amino acid sequence are shown in SEQ ID NO:4 and SEQ ID NO:5 respectively.The mouse GPR86 cDNA of SEQ ID NO:4 demonstrates the height homogeneity with people GPR86 (SEQ ID NO:2) sequence, and the amino acid sequence of mouse GPR86 (SEQ ID NO:5) demonstrates height homogeneity and similarity with people GPR86 (SEQ ID NO:3 and SEQ ID NO:7).

Therefore, people and mouse GPR86 are the members of g protein coupled receptor (GPCR) extended familys.

The GPR86 polypeptide

When being used for this paper, term " GPR86 polypeptide " means the polypeptide that comprises amino acid sequence shown in SEQ ID NO:3 or SEQ IDNO:5 or the SEQ ID NO:7, or its homolog, variant or derivant.Preferably, polypeptide comprises sequence shown in SEQ ID NO:3 or the SEQ ID NO:7, or its homolog, variant or derivant.Most preferably, polypeptide comprises sequence shown in the SEQ ID NO:7, or its homolog, variant or derivant.

" polypeptide " refers to comprise by peptide bond or modification peptide bond is peptide isostere two or more amino acid whose any peptides connected to one another or protein." polypeptide " both referred to be commonly referred to the short chain of peptide, oligopeptides or oligomer, also refer to be commonly referred to protein than long-chain.Polypeptide can comprise the amino acid beyond 20 kinds of gene coding amino acids.

" polypeptide " comprises by natural process, such as the processing of translation back, the perhaps amino acid sequence of modifying by chemical modification technology well-known in the art.Basic reader and more detailed monograph, and in the bundling research document good description has been carried out in this modification.Modification can occur in polypeptide Anywhere, comprises peptide backbone, amino acid side chain and amino or carboxyl terminal.The modification that should understand same type can identical or different degree be present in several sites of given polypeptide.And given polypeptide can comprise polytype modification.

Polypeptide can be owing to ubiquitination (ubiquitination) branch, and they also can be the ring-types that has or do not have branch.Can by translation back natural process or by synthetic method produce ring-type, branch with branch's annular polypeptide.Modification comprises acetylation; acidylate; the ADP-ribosylation; amidation; the covalent attachment of flavine; the covalent attachment of heme moiety; the covalent attachment of nucleotide or nucleotide derivative; the covalent attachment of lipid or lipid derivate; the covalent attachment of phosphatidylinositols; crosslinked; cyclisation; disulfide bond forms; demethylation; the formation of covalent cross-linking; the formation of cystine; the formation of pyroglutamic acid; formylation; the γ carboxylation; glycosylation; the GPI anchor forms; hydroxylation; iodate; methylate; the nutmeg acidylate; oxidation; proteolysis processing; phosphorylation; isoprenylation; racemic; selenonylization (selenoylation); sulfation; the amino acid of transfer RNA mediation adds on the protein such as arginylization and ubiquitination.Referring to for example Proteins-Structure and Molecular Properties, 2nd Ed., T.E.Creighton, W.H.Freeman and Company, New York, 1993 and Wold, F., Posttranslational Protein Modifications:Perspectives and Prospects, pgs.1-12 in Posttranslational Covalent Modification of Proteins, B.C.Johnson, Ed., Academic Press, New York, 1983; Seifter et al., " Analysis for proteinmodifications and nonprotein cofactors ", Meth Enzymol (1990) 182:626-646 and Rattan et al., " Protein Synthesis:Posttranslational Modifications and Aging ", AnnNY Acad Sci (1992) 663:48-62.

Term " variant ", " homolog ", " derivant " or " fragment " comprise when relevant with current file that (or a plurality of) of sequence amino acid whosely anyly substitute, variation, modify, displacement, deletion or add.Except as otherwise noted, otherwise just comprise this type of variant, homolog, derivant and the fragment of mentioning GPR86 when mentioning " GPR86 " and " GPR86 GPCR ".

Preferably, when being applied to GPR86, the gained amino acid sequence has the GPCR activity, more preferably has at least and the identical activity of GPR86 shown in SEQ ID NO:3 or SEQ ID NO:5 or the SEQ ID NO:7.Particularly, has the GPCR activity as if the gained amino acid sequence, then the homogeneity of term " homolog " covered structure and/or function aspects.With regard to sequence homogeneity (being similarity), preferably have at least 70%, more preferably at least 75%, more preferably at least 85% even more preferably at least 90% sequence homogeneity.At least 95%, more preferably at least 98% sequence homogeneity is more preferably arranged.These terms are also contained derived from the amino acid whose polypeptide that is GPR86 nucleotide sequence allelic variation.

When " receptor active " of mentioning acceptor such as GPR86 or " biologic activity ", these terms mean the metabolism or the physiological function of GPR86 acceptor, comprise the active of similar activity or improvement or those activity that reduce with undesired spinoff.The antigenicity and the immunogenicity activity that also comprise the GPR86 acceptor.These active methods of the example of GPCR activity and mensuration and quantification are known in this area, and also describe in detail in other place of presents.

When being used for this paper, " deletion " is defined as the variation that lacks one or more nucleotide or amino acid residue in nucleotide or the amino acid sequence respectively.When being used for this paper, " insertion " or " interpolation " is to cause in nucleotide or the amino acid sequence comparing with the material of natural generation, add respectively one or more nucleotide or amino acid residue variation.When being used for this paper, " substituting " is by replacing one or more nucleotide with different IPs thuja acid or amino acid respectively or amino acid causes.

GPR86 polypeptide described herein also can have deletion, the insertion that produces reticent variation and cause the amino acid residue of function equivalent amino acid sequence or substitute.Can carry out scrupulous amino acid replacement based on the similarity of polarity, electric charge, solubility, hydrophobicity, water wettability and/or the amphipathic characteristic aspect of residue.For example, electronegative amino acid comprises aspartic acid and glutamic acid; Positively charged amino acid comprises lysine and arginine; The amino acid that uncharged polar head group is arranged, has a similar hydrophilicity value comprises leucine, isoleucine, valine, glycocoll, alanine, asparagine, glutamine, serine, threonine, phenylalanine and tyrosine.

For example, can guard according to following table and substitute.In the secondary series same block, preferred the 3rd row can substitute each other with the amino acid in the delegation:

Aliphatics	Nonpolar	GAP
			ILV
		The polarity neutral		CSTM
	NQ
			The polarity zone electric charge	DE
	KR
		Aromatic series			HFWY

The GPR86 polypeptide also can comprise the allogeneic amino acid sequence, usually at N-end or C-end, preferably at the N end.Heterologous sequence can comprise to be influenced in the born of the same parents or the sequence (such as targeting sequencing) of exoprotein target.Heterologous sequence also can comprise the sequence that increases immunogenicity of polypeptides and/or be convenient to polypeptide evaluation, extraction and/or purifying.Another kind of particularly preferred heterologous sequence is the polyaminoacid sequence, such as preferred polyhistidine at the N-end.Especially preferably at least 10 amino acid, preferably at least 17 amino acid but be less than 50 amino acid whose polyhistidine sequences.

The GPR86 polypeptide can be the form of " maturation " protein, perhaps can be the part of larger protein such as fusion more.Comprise the sequence that contains secretion or targeting sequencing, former (pro-) sequence, help purifying such as the polyhistidine residue, it usually is favourable perhaps keeping the additional amino acid sequence of the appended sequence of stability during the recombinant production.

Can use known technology, by the favourable preparation GPR86 polypeptide of recombinant means.But also the technology that can use those of skill in the art to know prepares them by synthesizing mean such as solid phase synthesis.For example, in order to help to extract and purifying, form that also can fusion is produced this type of polypeptide.The example of fusion gametophyte comprises glutathione-S-transferase (GST), 6xHis, GAL4 (DNA combination and/or translation activation domain) and beta galactosidase.Comprising the proteolysis cleavage site to allow to remove the fusion sequence between fusion gametophyte and destination protein matter sequence, also is easily such as the fibrin ferment cleavage site.Preferably, the function that fusion can overslaugh destination protein matter sequence.

The GPR86 polypeptide can be the form of separating basically.This term means artificial change to state of nature.If " separation " composition or material exist at occurring in nature, it has just changed or has left its original environment so, and perhaps the two is furthermore.For example, natural polynucleotide, nucleic acid or the polypeptide that is present in the live animal is not " separation ", but is exactly " separation " with identical polynucleotide, nucleic acid or polypeptide that the coexisting substances of its state of nature separates, as the term that this paper adopted.

It should be understood that GPR86 protein can with the carrier or the mixing diluents of the intended purposes that does not hinder protein, and still regard as basically and to separate.This peptide species also can be the form of purifying basically, comprises protein in this case usually in preparation, wherein surpasses 90% in the preparation, and for example 95%, 98% or 99% protein is the GPR86 polypeptide.

This document also relates to the peptide that comprises a GPR86 polypeptide part.Therefore, comprise GPR86 fragment and homolog thereof, variant or derivant.The length of peptide can be between 2-200 amino acid, preferably between 4-40 amino acid.Peptide can be derived from GPR86 polypeptide disclosed herein, for example by digesting such as trypsase with suitable enzyme.Perhaps, can prepare peptide, fragment etc. by recombinant means or synthetic.

Term " peptide " comprises various synthetic peptide variation known in the art, such as retroinverso D peptide.Peptide can be antigenic determinant and/or t cell epitope.Peptide may be in vivo have immunogenic.Preferably, it can induce neutrality antibody in vivo.

Might determine from the GPR86 sequence of different plant species which district is (" homologous region ") guarded in the amino acid sequence, and which distinguishes change (" allos district ") between different plant species by comparison between different plant species.

Therefore, the GPR86 polypeptide can comprise the sequence corresponding to homologous region at least a portion.Homologous region demonstrates high homology at least between two species.For example, utilize test mentioned above, homologous region can show at least 70%, preferred at least 80%, more preferably at least 90% even more preferably at least 95% homogeneity at amino acid levels.Comprise the therapeutic strategy that the peptide corresponding to the sequence of homologous region can be used for hereinafter being described in more detail.Perhaps, the GPR86 peptide can comprise the sequence corresponding to allos district at least a portion.The allos district demonstrates low homology at least between two species.

GPR86 polynucleotide and nucleic acid

We have also described GPR86 polynucleotide, GPR86 nucleotide and GPR86 nucleic acid, their production method, purposes etc., have carried out more detailed description in other place of presents.

Term " GPR86 polynucleotide ", " GPR86 nucleotide " and " GPR86 nucleic acid " are used interchangeably, and mean the polynucleotide/nucleic acid or its homolog, variant or the derivant that comprise nucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4 or the SEQ ID NO:6.Preferably, polynucleotide/nucleic acid comprises SEQ ID NO:1 or SEQ ID NO:2, SEQ ID NO:6, the homolog of the most preferably nucleotide sequence of SEQ ID NO:2, or described nucleotide sequence, variant or derivant.

These terms also are intended to comprise can encode polypeptide described herein and/or peptide, the i.e. nucleotide sequence of GPR86 polypeptide.Therefore, GPR86 polynucleotide and nucleic acid comprise to encode and comprise the nucleotide sequence of the polypeptide of amino acid sequence shown in SEQ ID NO:3 or SEQ ID NO:5 or the SEQ ID NO:7, or its homolog, variant or derivant.Preferably, GPR86 polynucleotide and nucleic acid comprise to encode and comprise the nucleotide sequence of the polypeptide of amino acid sequence shown in the SEQ ID NO:7, or its homolog, variant or derivant.

" polynucleotide " are often referred to any polyribonucleotide or polydeoxyribonucleotide, can be RNA or the DNA or the modified RNA or the DNA of unmodified." polynucleotide " include but not limited to strand and double-stranded DNA, be strand and double stranded region potpourri DNA, strand and double-stranded RNA, be strand and double stranded region potpourri RNA, comprise can be strand or more typical be two strands or the DNA of strand and double stranded region potpourri and the heterocomplex of RNA.In addition, " polynucleotide " refer to comprise the two three sequences of RNA or DNA or RNA and DNA.The term polynucleotide also comprise DNA or the RNA that contains one or more modified bases, and main chain is for stability or other former thereby modify DNA or RNA." modified " base comprises for example tritylation (tritylated) base and uncommon base, such as inosine.DNA and RNA have been carried out multiple modification; Therefore, " polynucleotide " contain generally chemistry, enzyme or the metabolism improved form of the polynucleotide of finding at occurring in nature, and virus and the DNA of cells characteristic and the chemical species of RNA." polynucleotide " also contain the relatively short polynucleotide that usually are called oligonucleotides.

Those of skill in the art should be understood that because the degeneracy of genetic code, many nucleotide sequences identical polypeptide of encoding.

When being used for this paper, term " nucleotide sequence " refers to nucleotide sequence, oligonucleotide sequence, polynucleotide sequence and variant thereof, homolog, fragment and derivant (such as its part).Nucleotide sequence can be the DNA or the RNA of genome or synthetic or recombinant sources, and it can be double-stranded or represent strand or its combination of sense strand or antisense strand.Can use recombinant DNA technology (for example recombinant DNA) to prepare the term nucleotide sequence.

Preferably, term " nucleotide sequence " refers to DNA.

Term " variant ", " homolog ", " derivant " or " fragment " comprise that when being used for this paper any of (or a plurality of) nucleotide of sequence of GPR86 nucleotide sequence substitutes, makes a variation, modifies, replaces, deletes or add.Except as otherwise noted, otherwise just comprise this type of variant, homolog, derivant and the fragment of mentioning GPR86 when mentioning " GPR86 " and " GPR86 GPCR ".

Preferably, the nucleotide sequence coded polypeptide with GPCR activity of gained more preferably has and the identical activity of GPR86 shown in SEQ ID NO:3 or SEQ ID NO:5 or the SEQ ID NO:7 at least.Preferably, the homogeneity of term " homolog " intention covered structure and/or function aspects makes the polypeptide of the nucleotide sequence coded GPCR of the having activity of gained.With regard to sequence homogeneity (being similarity), preferably have at least 70%, more preferably at least 75%, more preferably at least 85%, more preferably at least 90% sequence homogeneity.At least 95%, more preferably at least 98% sequence homogeneity is more preferably arranged.The allelic variation of sequence also contained in these terms.

Sequence homology calculates

By any or a plurality of sequence and another sequence are carried out simply " eyeball " relatively (being rigorous comparison) to judge for example whether other sequence has at least 70% sequence homogeneity with this sequence, can measure the sequence homogeneity of any sequence shown in this paper.

Can utilize default parameter for example to calculate the computer program of the % homogeneity between two or more sequences with any algorithm that is suitable for measuring homogeneity by commercially available, also can measure relative sequence homogeneity.A representative instance of this computer program is CLUSTAL.Other computer program means of measuring homogeneity and similarity between two sequences is including, but not limited to GCG routine package (Devereux et al 1984 Nucleic Acids Research 12:387) and FASTA (Atschul et al1990 J Molec Biol 403-410).

Can calculate the % homology to continuous sequence, be about to a sequence and compare with another sequence, and with each amino acid in the sequence directly with another sequence in corresponding amino acid compare, compare a residue at every turn.This is called " non-notch " comparison.General only to carrying out this non-notch comparison than short number purpose residue relatively.

Though this is very simple and reliable method, but it is not considered for example in the identical pair of sequences of others, a comparison of inserting or deleting the amino acid residue that will make the back is lost shape, and therefore may cause when carrying out the integral body comparison, and the % homology reduces greatly.Therefore, most of sequence comparative approach are designed to produce best comparison under the situation of considering possible insertion and deletion, can excessively not punish whole homology score.Make local homology's maximization can realize this point by in sequence alignment, inserting " breach ".

But, these more complicated methods distribute " breach point penalty " for each breach that is present in the comparison, feasible same amino acid for similar number, sequence alignment with the least possible breach--reflects between two comparative sequences more high correlation is arranged--than the comparison with many breach, will obtain higher mark.Usually use " affine breach cost (affine gap cost) ", it sentences less point penalty with relative higher cost to each follow-up residue in the breach to the indentation, there that exists.This is the most frequently used breach points-scoring system.Certain high breach point penalty has generation the optimization comparison of less breach.Most of comparison programs allow to revise the breach point penalty.But, when utilizing this software to carry out the sequence comparison, preferably use default value.For example, utilizing GCG Wisconsin Bestfit when bag, the default breach point penalty of amino acid sequence is a breach-12 minute and each extension-4 minute.

Therefore, calculate maximum % homology and at first need under the situation of considering the breach point penalty, produce best comparison.The computer program that is suitable for implementing this comparison is GCG Wisconsin Bestfit bag (Universityof Wisconsin, U.S.A.; Devereux et al., 1984, Nucleic Acids Research 12:387).The example that can carry out sequence other software relatively is including, but not limited to BLAST bag (Ausubel et al., 1999 ibid-Chapter 18), FASTA (Atschul et al., 1990, J.Mol.Biol. is 403-410) with GENEWORKS compare tool group.BLAST and FASTA can both carry out off line and online searching (Ausubel et al., 1999 ibid, pages 7-58 to 7-60).

Though final % homology can be measured with regard to homogeneity, comparison process itself is not usually based on all or none paired comparisons.Usually change use amount table similarity into and get sub matrix (scaledsimilarity score matrix), it relatively distributes score for each according to chemical similarity or evolutionary distance in pairs.The example of this matrix commonly used is the BLOSUM62 matrix--the default matrix of blast program group.GCG Wisconsin program is used the public default value or the symbol comparison sheet of customization usually, if provide.For the GCG routine package, preferably use public default value; And in the situation of other software, preferably use default matrix, such as BLOSUM62.

Advantageously, adopt the BLAST algorithm, parameter is arranged to default value.Http:// www.ncbi.nih.gov/BLAST/blast_help.html describes the BLAST algorithm in detail, is collected herein by reference.Search parameter is defined as follows, the default parameter of being arranged to stipulate that can be favourable.

Advantageously, " substantive homogeneity " by BLAST evaluation is equal to the EXPECT value and is at least about 7, preferably at least about 9 and most preferably 10 or higher matching sequence.The default threshold value of EXPECT normally 10 in the blast search.

BLAST (the local comparison in basis research tool (Basic Local Alignment Search Tool)) is the heuristic search algorithm that program blastp, blastn, blastx, tblastn and tblastx are adopted; These programs belong to conspicuousness statistic law (Karlin and Altschul1990, the Proc.Natl.Acad.Sci.USA 87:2264-68 that utilizes Karlin and Altschul; Karlin and Altschul, 1993, Proc.Natl.Acad.Sci.USA 90:5873-7; Referring to Http:// www.ncbi.nih.gov/BLAST/Blast_help.html) and several improved judgements.Can revise blast program to carry out the sequence similarity search, for example identify the homolog of retrieve sequence.Discussion about the basic problem in the sequence library similarity searching can be referring to Altschul et al (1994) Nature Genetics 6:119-129.

Can implement following task: blastp-at protein sequence database comparing amino acid retrieve sequence at five blast programs that http://www.ncbi.nlm.nih.gov obtains; Blastn-compares the nucleotide retrieve sequence at nucleotide sequence database; Blastx-compares six frame conceptual translation products of nucleotide retrieve sequence (two chain) at protein sequence database; Tblastn-is at reading the nucleotide sequence database comparison protein retrieve sequence of dynamically translating (two chain) in the frame at all six; Tblastx-is at the relatively six frames translation of nucleotide retrieve sequence of six frames translation of nucleotide sequence database.

BLAST uses following search parameter:

HISTOGRAM (histogram)--show the histogram of each search score; Default is yes.(referring to the Parameter H in the BLAST handbook).

DESCRIPTIONS (description)--restriction reports to the short number of describing of the matching sequence of defined amount; Default restriction is 100 descriptions.(referring to the V parameter of man pages)

The significance,statistical threshold value of EXPECT (expection)--report and database sequence coupling; Default value is 10, makes according to the probabilistic model of Karlin and Altschul (1990), estimates only can find 10 couplings accidentally.If the significance,statistical that belongs to coupling just can not reported this coupling greater than the expection threshold value.Low expection threshold value is more rigorous, and the chance that causes reporting coupling still less.Can accept fractional value.(referring to the parameter E in the BLAST handbook).

CUTOFF (ending)--report high score section right by score.Default value is calculated by desired value (seeing above).Have only as the same with the significance,statistical that belongs to the isolated HSP that the score that equals cutoff is arranged at least HSP of report database sequence just when high of the significance,statistical that belongs to HSP.Higher cutoff is more rigorous, and the chance that causes reporting coupling still less.(referring to the parameter S in the BLAST handbook).Typically, can utilize EXPECT to handle the conspicuousness threshold value more intuitively.

ALIGNMENTS (comparison)--restricting data storehouse sequence is the number of the high score section of report of regulation to (HSP); Default restriction is 50.If the database sequence that surpasses this number happens to satisfy the significance,statistical threshold value (EXPECT and CUTOFF see above) of report, so just only report the coupling that belongs to maximum significance,statistical.(referring to the B parameter in the BLAST handbook).

MATRIX (matrix)--stipulated the alternately rating matrix of BLASTP, BLASTX, TBLASTN and TBLASTX.Default matrix is BLOSUM62 (Henikoff ﹠amp; Henikoff, 1992).Effectively alternative option comprises: PAM40, PAM120, PAM250 and IDENTITY (homogeneity).BLASTN does not replace rating matrix; Guiding MATRIX among the regulation BLASTN requires the reporting errors reaction.

STRAND (chain)--restricted T BLASTN is the top or the bottom chain of search database sequence only; Perhaps limit BLASTN, BLASTX or TBLASTX and only search for the top of retrieve sequence or the reading frame of bottom chain.

FILTER (filter)--shield in the retrieve sequence by Wootton ﹠amp; The SEG program of Federhen (1993) Computers and Chemistry 17:149-163 determines to have the low section of forming complicacy, perhaps by Claverie ﹠amp; The XNU program of States (1993) Computers and Chemistry 17:191-201 is determined, by the section that short period property inside repeats to form, for BLASTN, determine by the DUST program (referring to http://www.ncbi.nlm.nih.gov) of Tatusov and Lipman.Filtration can be exported from blast and be removed statistically evident but biologically insignificant report (for example hitting at common acidity, alkalescence or proline enrichment region) the result, stays the zone that has more biological significance that can be used in the retrieve sequence with the concrete coupling of database sequence.

In nucleotide sequence, in protein sequence, substitute the low-complexity sequence that filter is found with letter " X " (for example " XXXXXXXXX ") with letter " N " (for example " NNNNNNNNNNNN ").

Filter and only be only applicable to retrieve sequence (or its translation product), and be not used in database sequence.The default filtration of BLASTN is DUST, and the default filtration of other program is SEG.

During sequence in being applied to SWISS-PROT, SEG, XNU or both do not shield anything usually, therefore estimate to filter the fruit of can gross output coming into force.And, in some situation, the whole conductively-closed of sequence, this shows the significance,statistical that suspect at any coupling of not filtering the retrieve sequence report.

NCBI-gi--causes that NCBI gi identifier is also shown among the output result outside registration number and/or locus title.

Most preferably, the simple BLAST searching algorithm of utilizing http://www.ncbi.nlm.nih.gov/BLAST to provide is carried out sequence relatively.In some embodiment, when measuring sequence homogeneity, do not use the breach point penalty.

Hybridization

We also described can with sequence shown in this paper or its any fragment or derivant or with the nucleotide sequence of the complementary sequence hybridization of above-mentioned any sequence.

Hybridization refers to " process that nucleic acid chains combines by base pairing and complementary strand " (Coombs J (1994) Dictionary of Biotechnology, Stockton Press, New York NY), and as Dieffenbach CW and GS Dveksler (1995, PCR Primer, a Laboratory Manual, ColdSpring Harbor Press, Plainview NY) the described amplification procedure that in polymerase chain reaction technique, carries out.

Hybridization conditions is based on the melting temperature (Tm) of nucleic acid in conjunction with compound, as Berger and Kimmel (1987, Guide to Molecular Cloning Techniques, Methods in Enzymology, Vol 152, Academic Press, San Diego CA) instructed in, and given " the rigorous degree " of the qualification of hereinafter explaining.

Can be optionally with the nucleotide sequence of nucleotide sequence shown in this paper or its complementary sequence hybridization usually on the zone of at least 20, preferred at least 25 or 30, for example at least 40,60 or 100 or more continuous nucleotides with corresponding nucleotide sequence at least 70% shown in this paper, preferably at least 75%, more preferably at least 85% or 90% and even more preferably at least 95% or 98% homology.Preferred nucleotide sequence will comprise the zone with SEQ ID NO:1,2 or 4 homologies, preferably with one of sequence at least 70%, 80% or 90%, and more preferably at least 95% homology.

Term " optionally hybridization " refers to finding to use the nucleotide sequence as probe under the condition that target nucleotide sequences and probe are hybridized with the level that is significantly higher than background.Because for example have other nucleotide sequence in the cDNA that screens or the genomic DNA storehouse, so background hybridization may take place.In this case, background is inferred the signal level that is produced by the interaction between the non-specific DNA member in probe and the storehouse, and its strength ratio interacts low 10 times with the observed specificity of target DNA, preferably low 100 times.Interactional intensity can for example be used by for example radiolabeled probe ³²P measures.

The scope of presents also comprise can in by the time under the highest rigorous condition with the nucleotide sequence of nucleotide sequence hybridization shown in this paper.Hybridization conditions is based on the melting temperature (Tm) of nucleic acid in conjunction with compound, as Berger and Kimmel (1987, Guide to Molecular Cloning Techniques, Methods in Enzymology, Vol 152, Academic Press, San Diego CA) instructed in, and given " the rigorous degree " of the qualification of hereinafter explaining.

Maximum rigorous degree usually occurs in about Tm-5 ℃ (Tm than probe hangs down 5 ℃); High rigorous condition occurs in than low about 5-10 ℃ of Tm; Medium rigorous degree occurs in than low about 10-20 ℃ of Tm; And low rigorous degree occurs in than low about 20-25 ℃ of Tm.As those skilled in the art's understanding, maximum rigorous hybridization can be used for identifying or detecting identical nucleotide sequence, and medium (or low) rigorous hybridization can be used for identifying or detecting similar or relevant nucleotide sequence.

In a preferred embodiment, we disclose can be under rigorous condition (for example 65 ℃ and 0.1xSSC{1xSSC=0.15M NaCl, 0.015M trisodium citrate pH 7.0}) and the nucleotide sequence of one or more GPR86 nucleotide sequence hybridizations.When nucleotide sequence is two strands, contain in the doublet two chains of other or combination.When nucleotide sequence was strand, the complementary strand that should understand this nucleotide sequence was also included within the scope of presents.

We also described can with the complementary series of sequence shown in this paper or the nucleotide sequence of its any fragment or derivant hybridization.Same, also comprise can with the complementary nucleotide sequence of the sequence of sequence hybridization shown in this paper.Such nucleotide sequence is the example of nucleotide sequence variant.In this respect, term " variant " contain can with the complementary series of the sequence of nucleotide sequence hybridization shown in this paper.But preferably, term " variant " is contained can be under rigorous condition (for example 65 ℃ and 0.1xSSC{1xSSC=0.15M NaCl, 0.015M trisodium citrate pH 7.0}) and the complementary series of the sequence of nucleotide sequence hybridization shown in this paper.

The clone of GPR86 and homolog

We have described the nucleotide sequence with sequence shown in this paper or its any fragment or derivant complementation.If the fragment complementation of sequence shown in this sequence and this paper, this sequence just can be used as probe and is used for identifying and cloning similar GPCR sequence etc. other biosome so.

Therefore, our disclosing makes everybody can clone GPR86, its homolog and other structure or function related gene from people and other species such as mouse, pig, sheep etc.Be used for separating part or full-length cDNA and the genomic clone of the GPR86 that encodes with contained nucleotide sequence among SEQ ID NO:1, SEQID NO:2, the SEQ ID NO:4 or its fragment is identical or fully identical polynucleotide can be used as cDNA and genomic DNA hybridization probe from suitable storehouse.This probe also can be used for separating and the GPR86 gene has sequence similarity, the cDNA and the genomic clone of other gene of preferred heights sequence similarity (homolog and the straight gene to homolog that comprise the species of coding except that the people).Screening by hybridization, clone and sequencing technologies are that those skilled in the art are known, for example also are described among Sambrook etc. (seeing above).

Usually, the nucleotide sequence that is suitable as probe and indication sequence 70%, preferred 80%, more preferably 90% even more preferably 95% identical.Probe comprises at least 15 nucleotide usually.Preferably, this probe has at least 30 nucleotide, and can have at least 50 nucleotide.The preferred especially probe between 150-500 nucleotide, the probe of more preferred about 300 nucleotide.

In one embodiment, for the species beyond the people obtain coding GPR86 polypeptide, comprise homolog and straight polynucleotide to homolog, be included under the rigorous hybridization conditions and screen suitable storehouse, and separate part or full-length cDNA and the genomic clone that comprises described polynucleotide sequence with probe with SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4 or its fragment through mark.This hybridization technique is that those skilled in the art know.Rigorous hybridization conditions is exactly above to limit, or following condition: shear in the solution of salmon sperm dna in 42 ℃ and be incubated overnight containing 50% formamide, 5XSSC (150mM NaCl, 15mM trisodium citrate), 50mM sodium phosphate (pH7.6), 5XDenhardtShi solution, 10% sulfuric acid dextran and 20mg/ml sex change, in 0.1XSSC, wash filter membrane at about 65 ℃ then.

The functional examination method of GPR86

Can verify that the clone's infers the GPR86 polynucleotide by sequential analysis or functional examination method.For example, the GPR86 that can following mensuration infers or the receptor active of homolog.The secundum legem flow process is at external use RNA polymerase synthetic rna transcription thing that adds cap from the linearization plasmid template of coding GPR86 receptor cdna.The in-vitro transcription thing is suspended in the water, and final concentration is 0.2mg/ml.Extract the ovary leaf from the female toad that grows up, what obtain the V stage removes ovarian follicle (defolliculated) egg mother cell, and utilizes the microinjection instrument by injecting 50nl rna transcription thing to be expelled to (10ng/ egg mother cell) in the egg mother cell.Measure the electric current that individual African toad (Xenopus) egg mother cell response activator exposes with two electrode voltage pincers.Containing 115mM NaCl, 2.5mM KCl, 1.8mM CaCl ₂, 10mMNaOH-HEPES pH 7.2 standard medium in carry out record in room temperature.Africa toad system also can be used for screening activation part from known part and tissue/cell extract, as hereinafter in greater detail.

The expression determination method of GPR86

In order to design the therapeutic agent that can be used for treating the GPR86 relevant disease, the express spectra of measuring GPR86 is useful (no matter being wild type or specified mutant).Therefore, can determine that GPR86 expresses in which organ, tissue and cell type (and stage of development) with methods known in the art.For example, can implement traditional or " electronics " Northern (RNA blotting).Also can use reverse transcriptase PCR (RT-PCR) to measure the expression of GPR86 gene or mutant.The more sensitive method that is used to measure the GPR86 express spectra comprises RNA enzyme protection determination method known in the art.

It is to be used for detecting the laboratory technique that the genetic transcription thing exists that Northern analyzes, relate to that (Sambrook sees above, ch.7 and Ausubel with having combined film hybridization from the RNA of particular cell types or tissue through labeled nucleotide sequence, F.M.et al. sees above, ch.4 and 16).The similar computer technology (" electronics Northern ") of using BLAST is used in the identical or relevant molecule of search in nucleotide database such as GenBank or the LIFESEQ database (Incyte Pharmaceuticals).The advantage of such analysis is that they may be faster than the multiple crossing based on film.In addition, can change the sensitivity of computer search to determine that any specific coupling is included into identical (exact) or homology.

Polynucleotide described herein and polypeptide comprise that probe mentioned above can be used as research reagent and material is used to find animal and human's class treatment of diseases and diagnostic method/preparation, as explaining more in detail in other place of presents.

The GPR86 polypeptide expression

We also comprise the process that is used to produce the GPR86 polypeptide.This method is included in the host cell that (being the condition of expression of polypeptides) under the suitable condition cultivates the nucleic acid that comprises coding GPR86 polypeptide or its homolog, variant or derivant substantially.

In order to express the GPR86 of biologic activity,, promptly comprise the carrier of the necessary element of coded sequence of transcribing and translate insertion with the nucleotide sequence insertion suitable expression of coding GPR86 or its homolog, variant or derivant.

Can utilize the method for well known to a person skilled in the art to make up and comprise the sequence of the GPR86 that encodes and the suitable expression vector of transcribing and translate control element.These methods comprise extracorporeal recombinant DNA technology, synthetic technology and vivo gene reorganization.Sambrook, J. etc. (1989; Molecular Cloning, ALaboratory Manual, ch.4,8, and 16-17, Cold Spring Harbor Press, Plainview, N.Y.) and Ausubel, (1995 and periodic supplements such as F.M.; Current Protocols inMolecular Biology, ch.9,13, and 16, John Wiley ﹠amp; Sons, New York has described these technology in N.Y.).

Can use multiple expression vector/host system to comprise and express the coding GPR86 sequence.These include but not limited to microorganism, such as the bacterium that transforms with recombinant phage, plasmid or cosmid DNA expression vector; Yeast with the Yeast expression carrier conversion; Insect cell system with virus expression carrier (for example baculoviral) infection; With virus expression carrier (for example cauliflower mosaic virus (CaMV) or tobacco mosaic virus (TMV) (TMV)) or with bacterial expression vector (for example Ti or pBR322 plasmid) plant transformed cell system; Or zooblast system.It doesn't matter to use which kind of host cell.

" control element " or " adjusting sequence " is to interact with the non-translational region implementing to transcribe and translate (be enhancer, promoter and 5 ' and 3 ' non-translational region) with host cell proteins matter in the carrier.These elements can change aspect intensity and the specificity.According to employed carrier system and host, can use many suitable elements of transcribing and translate, comprise composition and inducible promoters.For example, when in bacterial system, cloning, can use inducible promoters, such as the BLUESCRIPT phasmid (Stratagene, La Jolla, Calif.) or the heterozygosis lacZ promoter of PSPORT1 plasmid (GIBCO/BRL) etc.Can in insect cell, use baculovirus polyhedrin body protein promoter.Can will be cloned in the carrier derived from promoter or the enhancer of vegetable cell genome (for example heat shock, RUBISCO and storage protein gene) or plant virus (for example viral promotors or targeting sequencing).In mammal cell line system, preferably from the promoter of mammalian genes or mammalian virus.If must produce the clone of the sequence of the coding GPR86 that comprises multicopy, can use carrier so based on SV40 or EBV with suitable selected marker.

In bacterial system, can select many expression vectors according to the desired use of GPR86.For example, when a large amount of GPR86 of needs induces antibody, the carrier of fusion high level expression that can the easy purifying of instruction.This carrier includes but not limited to multi-functional escherichia coli cloning and expression vector, such as BLUESCRIPT (Stratagene), wherein the sequence of coding GPR86 can be connected in the carrier and be in same reading frame to produce hybrid protein with amino terminal Met and follow-up 7 beta galactosidase residues; PIN carrier (Van Heeke, G.and S.M.Schuster (1989) J.Biol.Chem.264:5503-5509) or the like.(Promega, Madison Wis) also can be used for expressing the external polypeptide that forms fusion with glutathione S-transferase (GST) to the pGEX carrier.In general, this fusion is a solubility, and is easy to by being adsorbed onto on glutathione-sepharose 4B and carrying out wash-out purifying from the cell of dissolving subsequently and come out under having the condition of free glutathione.The protein for preparing in this system can be designed to comprise heparin, fibrin ferment or factor XA proteinase cutting site, thereby clone's desired polypeptides can arbitrarily partly be discharged from GST.

In saccharomyces cerevisiae, can use to comprise composition or inducible promoters, such as many carriers of the α factor, alcohol oxidase and PGH.Summary is referring to Ausubel (seeing above) and Grant etc. (1987; Methods Enzymol.153:516-544).

If the use plant expression vector can drive the expression of the sequence of coding GPR86 so with any in many promoters.For example, viral promotors can use or unite Ω targeting sequencing from TMV separately such as the 35S of CaMV and 19S promoter.(Takamatsu, N. (1987) EMBO are J.6:307-311.) or, can use plant promoter, such as small subunit or the heat shock promoter of RUBISCO.(Coruzzi, G.et al. (1984) EMBO is J.3:1671-1680; Broglie, R.et al. (1984) Science 224:838-843; And Winter, J.et al. (1991) Results Probl.Cell Differ.17:85-105.) can be by direct DNA transforms or pathogen mediates transfection with these construction introduced plant cells.In many common available summaries these technology have been described.(referring to for example Hobbs, S.or Murry, L.E.in McGraw Hill Yearbook of Science andTechnology (1992) McGraw Hill, New York, N.Y.; Pp.191-196.)

Can also use the insect system to express GPR86.For example, in a kind of such system, use autographa california nuclear polyhedrosis virus (Autographa californica nuclear polyhedrosisvirus) (AcNPV) to be used for expressing alien gene at fall army worm (Spodoptera frugiperda) cell or cabbage looper (Trichoplusia) larva as carrier.Can with the coding GPR86 sequence clone in the nonessential region of virus, such as polyhedron gene, and place under the control of polyhedrin promoter.The successful insertion of GPR86 will make the polyhedron gene inactivation and produce the recombinant virus that lacks coat protein.Recombinant virus can be used to infect for example fall army worm cell or cabbage looper larva then, wherein can express GPR86.(Engelhard，E.K.et al.(1994)Proc.Nat.Acad.Sci.91：3224-3227。)

In mammalian host cell, can use many expression systems based on virus.If use adenovirus, just the sequence of coding GPR86 can be connected to the adenovirus that comprises late promoter and tripartite leader[and transcribe/translate in the compound as expression vector.Inserting virus genomic nonessential E1 or E3 district can be used for obtaining expressing the survived virus of GPR86 in infected host cell.(Logan, J.and T.Shenk (1984) Proc.Natl.Acad.Sci.81:3655-3659.) in addition, can use transcriptional enhancer, increases expression in the mammalian host cell such as Rous sarcoma virus (RSV) enhancer.

Therefore, can in the dhfr Chinese hamster ovary celI of for example human embryo kidney (HEK) 293 (HEK293) cell or adhesion, express the GPR86 acceptor.In order to make expression of receptor maximization, from receptor cdna, remove whole 5 ' and 3 ' non-translational region (UTR) usually, insert pCDN or pCDNA3 carrier then., and under the situation that has 400mg/ml G418, select with each receptor cdna transfectional cell by the fat transfection.After selecting for 3 weeks, select indivedual clones, and enlarged culture is done further to analyze.Only use the HEK293 of carrier transfection or Chinese hamster ovary celI as negative control.Express the clone of each acceptor for separating stable, select about 24 to clone and analyze usually by the Northern engram analysis.Usually about 50% analyze in the G418 resistance clone and can detect receptor mrna.

Can also use human artificial chromosome (HAC) to deliver the dna fragmentation bigger than the DNA that in plasmid, comprises and express.For therapeutic purposes, make up the HAC of about 6kb-10Mb, and deliver by conventional delivering method (liposome, polycation amino polymer or vesica).

Can also be with realize the encoding more efficient translation of sequence of GPR86 of special start signal.Such signal comprises ATG initiation codon and flanking sequence.The sequence of GPR86 and initiation codon thereof and upstream sequence insert suitable expression if will encode, and may not need additional transcription or translation control signal so.But, iff having inserted coded sequence or its fragment, so just should provide external source translation control signal, comprise the ATG initiation codon.And initiation codon should be to guarantee the translation of whole embolus in correct reading frame.External source translation element and initiation codon can be multiple sources, and natural and synthetic can.By comprising the enhancer that is suitable for used specific cells system,, can strengthen expression efficiency (Scharf, D.et al. (1994) ResultsProbl.Cell Differ.20:125-162) such as described in the document.

In addition, can regulate ability that the institute insetion sequence expresses or select host cell strain according to it with the ability that the expection mode is processed expressed protein.Described peptide modified acetylation, carboxylation, glycosylation, phosphorylation, fatization and the acylation of including but not limited to.Processing promotes correct insertion, folding and/or function after can also using the translation of scinderin matter " preceding former (prepro) " form.From American type culture collection (ATCC, Bethesda, Md) can obtain to have the specific cells structure of translation back processing and the different host cells (for example CHO, HeLa, MDCK, HEK293 and WI38) of characteristic mechanism, can select them to guarantee the correct modification and the processing of foreign protein.

For production recombinant protein long-term, high yield, preferred stable expression.For example, can utilize following expression vector transform can stably express GPR86 clone, described expression vector can comprise virus replication starting point and/or endogenous Expression element and selectable marker gene on identical or the carrier that separates.After introducing carrier, can allow cell in rich medium, to grow about 1-2 days, change the selection nutrient culture media then into.The purpose of selected marker is to give the selection resistance, and its existence allows to cultivate and reclaim the cell of successful expression institute calling sequence.Can utilize the increase resistance clone of stable transformed cells of the tissue culture technique that is suitable for this cell type.

Many selective systems can be used to reclaim transformation cell lines.These include but not limited to can be used for respectively the herpes simplex virus thymidine kinase gene (Wigler of tk-or apr-cell, M.et al. (1977) Cell 11:223-32) and adenine phosphoribosyl transferase gene (Lowy, I.et al. (1980) Cell 22:817-23).Antimetabolite, microbiotic or Herbicid resistant can be used as the basis of selection equally.For example, dhfr gives methopterin resistance (Wigler, M.et al. (1980) Proc.Natl.Acad.Sci.77:3567-70); Npt gives aminoglycoside neomycin and G-418 resistance (Colbere-Garapin, F.et al (1981) J.Mol.Biol.150:1-14); Reach als or pat and give chlorsulfuron and phosphinotricin acetyltransferase resistance (Murry sees above) respectively.Describe other and selected gene, for example allowed cell to utilize indoles to replace the trpB of tryptophane, perhaps allowed cell to utilize histidinol to replace the hisD of histidine.(Hartman, S.C.and R.C.Mulligan (1988) Proc.Natl.Acad.Sci.85:8047-51.) nearest, utilize this witness marking to obtain to popularize as anthocyanin, beta-Glucuronidase and substrate GUS thereof and luciferase and substrate luciferin thereof.These marks not only can be used for identifying transformant, also can be used for quantizing to be attributable to the instantaneous or stable protein expression of specific support system.(Rhodes，C.A.et al.(1995)Methods Mol.Biol.55：121-131.)

Though whether the existence that marker gene is expressed hints and also has genes of interest that the existence of this gene and expression also need to confirm.For example, if the sequence of the GPR86 that will encode is inserted in the marker gene sequence, can identify the transformant of the sequence that comprises the GPR86 that encodes by lacking marker gene function so.Perhaps, marker gene can be connected with the sequence of coding GPR86 under being in the control of single promoter.Marker gene responds the expression that the expression of inducing or selecting also shows tandem gene usually.

Perhaps, can identify nucleotide sequence that comprises the GPR86 that encodes and the host cell of expressing GPR86 by the known multiple flow process of those skilled in the art.These flow processs include but not limited to be used to detect and/or quantize DNA--DNA or DNA--RNA hybridization and the protein biologicall test or the immunoassay of nucleic acid or protein sequence, and they comprise the technology based on film, solution or chip.

Utilize the fragment of polynucleotide of probe or fragment or coding GPR86, can detect the existence of the polynucleotide sequence of the GPR86 that encodes by DNA--DNA or DNA--RNA hybridization or amplification.Determination method based on nucleic acid amplification relates to utilization based on the oligonucleotides of the sequence of coding GPR86 or the transformant that oligomer detects the DNA or the RNA that comprise the GPR86 that encodes.

This area knows that utilization detects and measure the several different methods that GPR86 expresses to protein special polyclone or monoclonal antibody.The example of these technology comprises enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) and fluorescence activated cell letter sorting (FACS).Preferred utilize to last two the non-interfering epi-positions of GPR86 have that reactive monoclonal antibody carries out based on two sites, based on monoclonal immunoassay, but can use the competitive binding assay method.This area is described these determination methods and other determination method in detail, for example Hampton R. etc. (1990; Serological Methods, aLaboratory Manual, Section IV, APS Press, St Paul, Minn.) and Maddox, D.E. etc. (1983; J.Exp.Med.158:1211-1216).

Those skilled in the art know multiple label and conjugation techniques, and they can be used for various nucleic acid and determined amino acid method.The means through mark hybridization or PCR mark that are used to produce the correlated series of the polynucleotide that are suitable for detecting coding GPR86 comprise oligomer mark (oligolabeling), nick translation (nick translation), end mark or utilize the pcr amplification that carries out through labeled nucleotide.Perhaps, can be with the coding sequence of GPR86 or its any fragment cloning to the carrier that is used for producing the mRNA probe.This carrier is known in this area, can buy from the market, and by adding suitable RNA polymerase such as T7, T3 or SP6 and through labeled nucleotide, can being used at the vitro synthesized RNA probe.Utilize commercially available plurality of reagents box, such as Pharmacia ﹠amp; Upjohn (Kalamazoo, Mich.), (Madison, Wis.) (Cleveland, those kits that Ohio) provide can be implemented these flow processs to Promega with U.S.Biochemical Corp..The suitable reporter molecules or the label that can be used for detecting easily comprise radioactive nuclide, enzyme, fluorescer, chemiluminescence agent or developer, and substrate, accessory factor, inhibitor, magnetic-particle or the like.

Can cultivate nucleotide sequence transformed host cells being suitable for marking protein and from cell culture, reclaiming under the condition of protein with coding GPR86.The protein that is produced by transformant can be arranged in cell membrane, secretes away or is included in the cell, and this depends on the sequence and/or the carrier of use.Those skilled in the art should be understood that and the expression vector that comprises the polynucleotide of the GPR86 that encodes can be designed to comprise the burst that instructs the GPR86 secretion to pass through protokaryon or eukaryotic cell membrane.Other structure can be used for the sequence of coding GPR86 is connected the nucleotide sequence in the following polypeptide structure of coding territory, and described polypeptide domain is with the purifying of convenient soluble protein.This purifying convenient structure territory includes but not limited to metal chelating peptide, such as allow at the histidine that carries out purifying on the immobilization metal-tryptophane assembly (modules), allow on the immobilization immunoglobulin (Ig), to carry out the albumin A domain of purifying, and at (the Immunex Corp. of FLAGS extension/protein affinity purification system, Seattle, Wash.) the middle domain that uses.Comprise the joint sequence that can cut between purification structure territory and GPR86 coded sequence, (Invitrogen, San Diego Calif.) can be used for convenient purifying such as factor XA or those special joint sequences of enterokinase.A kind of such expression vector is expressed fused protein that comprises GPR86 and the nucleic acid of encoding 6 histidine residues, and the nucleic acid of 6 histidine residues of wherein encoding is before thioredoxin or enterokinase cleavage site.The histidine residues facility is at immobilized metal ion affinity chromatography (IMIAC; See Porath, J.et al. (1992) Prot.Exp.Purif.3:263-281) on purifying, and the enterokinase cleavage site provides from the means of fusion purifying GPR86.Kroll, D.J.et al. (1993; Discussion about the carrier that comprises fusion is provided DNA Cell Biol.12:441-453).

Not only can produce the GPR86 fragment, also can synthesize and produce the GPR86 fragment by the direct peptide that uses solid phase technique by recombinant production.(Merrifield J. (1963) J.Am.Chem.Soc.85:2149-2154.) can implement protein synthesis by artificial technology or robotization mode.For example utilize Applied Biosystems 431A peptide synthesizer (Perkin Elmer) can realize that robotization is synthetic.Can separately synthesize various GPR86 fragments, then their be made up to produce full-length molecule.

Biology sensor

GPR86 polypeptide, nucleic acid, probe, antibody, expression vector and part can be used as (with being used for production) biology sensor.

According to Aizawa (1988), Anal.Chem.Symp.17:683, biology sensor is defined as the acceptor that is used for molecular recognition, and for example the selection layer of immobilized antibody or acceptor such as GPR86 g protein coupled receptor and being used to transmits the unique combination of the forwarder of measured value.One group of such biology sensor will detect the change of the top layer optical property that the interaction owing to acceptor and surrounding medium causes.In this technology, especially to mention oval symmetry (ellipso-metry) and surperficial excimer resonance (surface plasmonresonance).The biology sensor that has mixed GPR86 can be used for detecting the GPR86 part, for example the existence of the analog of nucleotide such as purine or purine analogue or these parts or level.The structure of this biology sensor is well known in the art.

Therefore, the clone of expressing the GPR86 acceptor can be used as reporting system and is used for detector ligand, promotes the ATP or other second messenger (Watt et al., 1998, the J Biol Chem May 29 that form such as the acceptor via [3H] inositol monophosphate; 273 (22): 14053-8).Hoffman et al., 2000, Proc Natl AcadSci U S A Oct 10; 97 (21): also described the receptor-ligand biology sensor among the 11215-20.Comprise the optics of GPR86 and other biology sensor and also can be used for detecting level or existence with G albumen and other protein interactions, Figler et al for example, 1997, Biochemistry Dec 23; 36 (51): 16288-99 and Sarrio et al., 2000, Mol Cell Biol 2000 Jul; 20 (14): described in the 5164-74.For example, US 5,492, described the sensor element of biology sensor in 840.

The Screening test method

No matter be GPR86 polypeptide natural or reorganization, comprise the process that homolog, variant and derivant all can be used for screening bind receptor and activation GPR86 (activator) or suppress the compound of GPR86 activation (antagonist).

Therefore, polypeptide also can be used for the combination of evaluation Example such as cell, acellular goods, chemicals library and medium and small molecule substrate of natural product mixture and part.These substrates and part can be natural substrate and part, perhaps can be structure or functional simulation thing.Referring to Coligan et al., Current Protocols inImmunology 1 (2): Chapter 5 (1991).

The GPR86 polypeptide is responsible for many biological functions, comprises many pathology, those that describe in all " GPR86 relevant disease " as mentioned.Therefore, thirst for to find to stimulate GPR86 on the one hand and the compound and the medicine that can suppress the GPR86 function on the other hand.Usually, activator and antagonist can be used for treatment and prevention such as dopamine relevant disease such as Parkinson's disease, heart disease such as supraventricular or ventricular arrhythmia, low blood pressure, feel sick, tourette syndrome, stress with situation such as pain.

Activator can activate the GPR86 acceptor to any degree.Similarly, but antagonist deactivation or suppress GPR86 and be activated to any degree.Therefore, any degree of available antagonist (partial antagonist) part deactivation GPR86 acceptor, basis intrinsic to it or background activity level or complete inactivation are to this level (antagonist or antagonist) fully.Antagonist even also can make the acceptor deactivation to odd jobs (inverse agonists) for example.Therefore, term " antagonist " specifically comprises complete antagonist, partial antagonist and inverse agonists.

Term " activator " and " antagonist " also are included in transcribes and/or translation skill is regulated those molecules that GPR86 expresses, and those molecules of regulating the GPR86 activity.

Can according to the structural research appropriate design of peptide molecule shape might with the candidate compound of GPR86 protein interaction.Be used for determining which site and a kind of means of specific other protein interaction are physical arrangement mensuration, for example X-ray crystallography or two dimensional NMR techniques.These will provide guidance for which amino acid residue forms the molecule contact region.Can be about the detailed description that protein structure is measured referring to for example Blundell and Johnson (1976) Protein Crystallography, AcademicPress, New York.

The replacement method of appropriate design is to use screening process, and it relates generally to allow GPR86 contact candidate's instrumentality and detects its effect.Usually, measuring method comprises producing chooses the suitable cell of expressing the GPR86 receptor polypeptides with gametophyte protein in its surface wantonly, allows GPR86 or cell or both contact candidate's instrumentality, and detects the interior level variation of born of the same parents of correlation molecule.

Its concentration is subjected to the GPCR activity, and particularly GPR86 activity influence and the molecule that can be used as the active mark of GPR86 are known in this area.For convenience's sake these molecules are called " GPCR sensitivity label thing ".The example of this GPCR sensitivity label thing comprises cellular calcium level, calcium current amount, adenylate cyclase enzyme level and cyclic adenosine monophosphate level.In a preferred embodiment, GPCR sensitivity label thing comprises cyclic adenosine monophosphate (cAMP) in the born of the same parents, and screening comprises the variation that detects cyclic adenosine monophosphate level in the born of the same parents.

In particularly preferred embodiments, can under the situation that has gametophyte protein, screen GPR86; This gametophyte protein can be with GPR86 receptor polypeptides coexpression, preferably in born of the same parents.Gametophyte protein preferably can comprise G albumen, and such as the G albumen of GPR86 preference, Gi or the pungency G albumen that mixes are such as G _{α 16}

Carry in the screening of G Protein G i of GPR86 and GPR86 preference in use, activator reduces cAMP concentration level in the born of the same parents, and antagonist improves cAMP concentration in the born of the same parents.

At the pungency G albumen that uses GPR86 and mix such as G _{α 16}Screening in, activator improves cAMP concentration level in the born of the same parents, and antagonist reduces cAMP concentration in the born of the same parents.

Therefore, we disclose the method for identifying the GPR86 activator with the G Protein G i coupling of GPR86 preference the time, and this method comprises that whether the level that makes the cells contacting candidate compound of expressing the GPR86 acceptor and measure cyclic adenosine monophosphate in the described cell (cAMP) is owing to described contact reduces.Perhaps, GPR86 can with the pungency G albumen that mixes such as G _{α 16}Coexpression, so this method can comprise making and expresses GPR86 and G _{α 16}The cells contacting candidate compound and the level of measuring cyclic adenosine monophosphate in the described cell whether owing to described contact improves.

We also disclose the method for identifying the GPR86 antagonist with the G Protein G i coupling of GPR86 preference the time, and this method comprises that whether the level that makes the cells contacting candidate compound of expressing the GPR86 acceptor and measure cyclic adenosine monophosphate in the described cell (cAMP) is owing to described contact improves.Perhaps, if GPR86 and the pungency G albumen that mixes such as G _{α 16}Coexpression makes so and expresses GPR86 and G _{α 16}The cells contacting candidate compound and the level of measuring cyclic adenosine monophosphate in the described cell whether owing to described contact reduces.

The cell that can be used for screening can be all kinds.This type of cell comprises from animal, yeast, fruit bat or colibacillary cell.Allow then expressed receptor the cell cell membrane of expressed acceptor (or comprise) engaged test compound with observe in conjunction with or to the stimulation or the inhibition of functional response.For example, can give Africa xenopus egg mother cell injection GPR86 mRNA or polypeptide as other place in greater detail, and measure by being exposed to the electric current that test compounds is induced with voltage clamp.

And, can use little physiometry determination method (microphysiometric assay) to measure the GPR86 receptor active.The activation of multiple second messenger system causes spraying small amount of acid from cell.The acid that forms mainly is the result who handles the metabolic activity raising that energy demand is provided to intracellular signal.It is very little changing around the pH of the nutrient culture media of cell, but can (Molecular Devices Ltd., Menlo Park Calif.) detects by the little physiometry instrument of CYTOSENSOR for example.Therefore, CYTOSENSOR can detect and utilize the acceptor of the intracellular signal approach coupling of energy, the activation of all g protein coupled receptors as described herein.

Except testing every kind of candidate compound one by one, advantageously produce and screen the candidate ligand library with the GPR86 acceptor.Therefore, for example, assembled above 200 kinds of storehouses of inferring receptors ligand and be used for screening.This storehouse comprises: mediator, hormone and knownly stride the chemotactic factor (CF) that film (7TM) acceptor works for seven times via the people; May be people 7TM acceptor, not identify the natural generation compound of inferring activator of the nonmammalian biologic activity peptide of its mammal homologue as yet; Find at occurring in nature, but with the compound of unknown native ligand activation 7TM acceptor.Utilize other place functional examination method (being calcium, cAMP, little physiometry instrument, egg mother cell electrophysiology etc.) and binding assay in greater detail, can screen the acceptor of known ligand with this storehouse referring to other place.But, have many also relevant so far mammalian receptors that activate parts (activator) or deactivation part (antagonist).Therefore, the active ligand of these acceptors may not be included in the part storehouse of identifying up to now.Therefore, also on function, tissue extract is screened GPR86 acceptor (utilizing functional screening methods such as calcium, cAMP, little physiometry instrument, egg mother cell electrophysiology) to identify native ligand.The extract that produces positive functional response can be segmented continuously, and each fraction of mensuration as described herein, up to separating and identifying the activation part.

Being presented at the 7TM acceptor of expressing in HEK 293 cells stimulates with PLC activation and calcium mobilization and/or cAMP on function or suppresses coupling.Therefore, a kind of triage techniques is included in and utilizes the cell of expressing GPR86 acceptor Africa xenopus egg mother cell, CHO or the HEK293 cell of transfection (for example through) in the system that pH in the outer or born of the same parents of born of the same parents that measurement causes by receptor activation or cellular calcium change.In this technology, can allow compound contact the cell of expressed receptor polypeptide.Measure second messenger's reaction then, for example signal transduction, pH change or whether the variation of calcium level activates with definite potential compound or suppress acceptor.

In experiments of measuring, observe in HEK 293 cells, in the acceptor transfectional cell or the basic calcium level in the vehicle Control cell be in the normal range of 100nM-200nM.Load HEK 293 cells of expressing GPR86 or reorganization GPR86 with fura 2, and in one day, assess the calcium mobilization of agonist induction surpassing 150 selected parts or tissue/cell extract.Similarly, stimulation or the inhibition that utilizes standard cAMP quantitative determination process that the HEK 293 cells assessment cAMP that expresses GPR86 or reorganization GPR86 is produced.The activator that test presents calcium transient phenomenon or cAMP fluctuation in the vehicle Control cell is to determine that whether this reaction is that the transfectional cell of expressed receptor is distinctive.

Another kind method relates to by measuring the inhibition that the receptor-mediated cAMP of GPR86 and/or adenyl cyclase are gathered or stimulating screens receptor inhibitor.These class methods relate to the acceptor transfecting eukaryotic cells with at the cell surface expression acceptor.Exist under the situation of acceptor cellular exposure in potential antagonist then.Measure the accumulated amount of cAMP then.If therefore the potential antagonist bind receptor also suppresses receptors bind, so receptor-mediated cAMP or adenylate cyclase activity level will reduce or improve.

The another kind of method that detects GPR86 receptor stimulating agent or antagonist is a United States Patent (USP) 5,482, and the technology of describing in 835 based on yeast is collected herein by reference.

When candidate compound is a protein, particularly when antibody or peptide, can utilize display technique of bacteriophage to screen the candidate compound library.Phage display is a molecular screening scheme of utilizing recombinant phage.This technology relates to encoding does not have the genetic transformation bacteriophage of a kind of compound in storehouse from candidate compound, make each bacteriophage or phasmid express a kind of specific candidate compound.Bacteriophage (preferably being fixed on the solid support) through transforming is expressed suitable candidate compound and is illustrated on their phage ghost.Can be by selection strategy enrichment in conjunction with the specificity candidate compound of GPR86 polypeptide or peptide based on affine interaction.Identify successful candidate's preparation then.Phage display has the advantage that is better than standard affinity ligand triage techniques.Phage surface with to the tightr similar 3-d modelling show candidate preparation of natural occurred conformation of candidate's preparation.This provides more special and higher affinity combination for realizing the screening purpose.

The method utilization in the another kind of screening compounds storehouse eucaryon or the prokaryotic host cell of the recombinant DNA molecules stable conversion of expressing compound library.Live or machine made this type of cell all can be used for standard binding partners determination method.Also can be referring to Parce et al. (1989) Science 246:243-247; With Owicki etal. (1990) Proc.Nat ' l Acad.Sci.USA 87:4007-4011, they have described the sensitive method that detects cell effect.Competitive assays is particularly useful, wherein makes the known labelled antibody in conjunction with the GPR86 polypeptide of cells contacting of expressing compound library, such as ¹²⁵I-antibody and specimen are such as just measuring it to the candidate compound of the binding affinity of binding compositions or insulation together.The binding partners of the polypeptide of process mark separating and combining and free is to estimate combination degree then.The binding capacity of specimen is inversely proportional to the amount of the labelled antibody that combines polypeptide.

Can use any the binding partners in many technology to separate to estimate combination degree with free with combination.Separating step can relate to the flow process of washing, be adsorbed onto on the plastics washing then or centrifuge cell film such as being adsorbed onto on the filter membrane then usually.

Also have a kind of method to use the polypeptide or the peptide of the purifying of dissolving, unpurified or dissolving, for example from the eucaryon that transforms or prokaryotic host cell, extract.This for " molecule " binding assay provide the specificity raising, can robotization and the advantage of high flux medicine inspection.

The technology of another kind of screening candidate compound relates to provides high flux screening to have suitable binding affinity, the method that for example the GPR86 polypeptide is had the noval chemical compound of suitable binding affinity has a detailed description in disclosed International Patent Application WO 84/03564 on September 13rd, 1984 (Commonwealth Serum Labs.).At first, at solid substrate, for example synthesize a large amount of different little peptide test compounds on plastics pin or some other the suitable surface; Referring to Fodor et al. (1991).Allow the polypeptide of all pins and dissolving react then and wash.Next step relates to the polypeptide that detects combination.Therefore identify and the interactional compound of polypeptid specificity.

The part binding assay provides definite acceptor pharmacological direct method, and can adapt to high throughput format.The part of acceptor that can the radioactive label purifying to height ratio is lived (50-2000Ci/mmol) to carry out combination research.Determine that then the radioactive label process does not reduce the activity of part to its acceptor.Optimize damping fluid, ion, pH and other instrumentality, such as the condition determination of nucleotide to determine to be suitable for the feasible signal to noise ratio (S/N ratio) in film and full cell receptor source.For these determination methods, specific receptor deducts the radioactivity of measuring when having excessive unmarked competitive part in conjunction with being defined as total binding radioactivity.When possible, availablely surpass a kind of competitive part and define remaining non-specific binding.

Determination method can only be tested the combination of candidate compound, wherein by the means of the direct or indirect label that combines with candidate compound or the adhesion that the cell of acceptor is carried in detection in the determination method of the competition that relates to mark competition thing.In addition, utilize the detection system of the cell be suitable for carrying on the surface acceptor, these determination methods can be tested candidate compound and whether be caused the signal that produced by receptor activation.Usually there is the mortifier of measuring activation under the situation of known activator, and observing the influence of the activation that the candidate compound that exists causes activator.

In addition, this mensuration can only comprise mixes candidate compound to form potpourri with the solution that contains the GPR86 polypeptide, measure the GPR86 activity in the potpourri, and with the active step that compares with standard of the GPR86 of potpourri.

Also can dispose determination method, be used for detecting the compound pair cell GPR86 mRNA of interpolation and the influence of protein production with the antibody of GPR86 cDNA, protein and protein.For example, can utilize monoclonal and polyclonal antibody, make up ELISA by standard method known in the art and measure the secretion level of GPR86 protein or cell, and this can be used for finding suppressing or strengthen the preparation (being also referred to as antagonist or activator respectively) by the cell or tissue generation GPR86 of proper handling in conjunction with level.This area is implemented the standard method of Screening test method as everyone knows.

The example of potential GPR86 antagonist comprises antibody, perhaps in some situation, comprise nucleotide and analog thereof, comprise purine and purine analogue, with closely-related oligonucleotides of part or the protein of GPR86, the fragment of part for example, thereby perhaps bind receptor but the micromolecule of the prevention receptor active that do not induce reaction.

Therefore, we also provide can specificity in conjunction with the compound of GPR86 polypeptide and/or peptide.

Term " compound " refers to chemical compound (natural generation or synthetic), such as biology macromolecule (for example nucleic acid, protein, non-peptide or organic molecule) or by extract or even the inorganic elements or the molecule of biomaterial such as bacterium, plant, fungi or animal (particularly mammal) cell or tissue preparation.Preferred compound is an antibody.

Implementing this type of screens necessary material and can be packaged in the screening reagent box.This type of screening reagent box can be used for identifying the activator, antagonist, part, acceptor, substrate, enzyme of GPR86 polypeptide etc., or reduces or strengthen the compound that the GPR86 polypeptide is produced.The screening reagent box comprises: (a) GPR86 polypeptide; (b) recombinant cell of expression GPR86 polypeptide; (c) cell membrane of expression GPR86 polypeptide; Or (d) antibody of GPR86 polypeptide.The screening reagent box can be chosen wantonly and comprise operation instructions.

Transgenic animals

We also disclose can express normal level, compare with the normal expression level and to raise or the transgenic animals of natural or reorganization GPR86 or homolog, variant or the derivant of reduction level.Preferably, these type of transgenic animals are inhuman mammals, such as pig, sheep or rodent.Most preferably, transgenic animals are mouse or rat.

We disclose wherein whole or the natural GPR86 gene of part is used the transgenic animals of replacing from the GPR86 sequence of another biosome.Preferably, this biosome is another species, and optimum is chosen.In highly preferred embodiment, we disclose the mouse that its whole basically GPR86 gene personnel selection GPR86 gene is replaced.These type of transgenic animals, and the animal with wild type GPR86 can be used for screening activator and/or the antagonist of GPR86.

For example, this type of determination method can relate to wild type animal or transgenic animals, or its part, and cell, tissue or the organ of preferred transgenic animals are exposed to candidate substances, and measures the relevant phenotype of GPR86, such as pain or inflammation.Can also implement to use derived from the cell of relevant animal and measure screening based on cell to the influence of cyclic adenosine monophosphate concentration in the born of the same parents.

We also disclose the transgenic animals that comprise the GPR86 gene that function destroyed, and wherein any one or the multinomial function of disclosed GPR86 suffered partially or completely to eliminate in the presents.Comprise because one of the GPR86 gene or the sudden change of multinomial afunction, comprise deletion and the transgenic animals of expressive function GPR86 (" GPR86 knock-out animal ") not.

Compare with the wild type animal, the transgenic animals (GPR86 knock-out animal) that lack functional GPR86 demonstrate the neurological susceptibility of change to pain.Particularly, the GPR86 knock-out animal is more insensitive to pain.And the GPR86 knock-out animal demonstrates the neurological susceptibility of change to inflammatory pain, and particularly the neurological susceptibility to inflammatory pain reduces.

Also comprise the partial loss of function mutant, for example may have the incomplete knock-out animal of deletion in the selected part of GPR86 gene.Can be by optionally replacing or delete the relevant portion of GPR86 sequence, for example important protein matter domain produces this animal on the function.

This mutant of afunction wholly or in part can be used as the model of GPR86 relevant disease, particularly pain or inflammatory disease.The animal of display part afunction can be exposed to candidate substances and strengthen phenotype, promptly strengthen the material that (in the situation of GPR86) observed pain sensitivity reduces phenotype to identify.Utilize other local method of identifying of presents, can also detect other parameter, such as the variation of cyclic adenosine monophosphate level in the born of the same parents.

Part and complete knock-out animal also can be used for identifying selective agonist and/or the antagonist of GPR86.For example, use activator and/or antagonist can for wild type and GPR86 deficient animals (knock-out animal).The selective agonist or the antagonist that it will be appreciated that GPR86 are influential to the wild type animal, but to the not influence of GPR86 deficient animals.Particularly, design special determination method, determine whether it produces physiological reaction when lacking GPR86 to assess potential medicine (candidate ligand or compound).This can be by giving aforesaid transgenic animals drug administration, and the specific reaction of measuring animal then realizes.Also can implement to use derived from the cell of relevant animal and measure to the influence of cyclic adenosine monophosphate concentration in the born of the same parents similarly based on the method for cell.This type of animal also can be used for testing the effect of the medicine that comes out by the Screening and Identification of presents description.

In another embodiment, use transgenic animals to screen with partial loss of function phenotype.In such embodiment, screening can relate to be measured partially or completely recovering or being returned to the wild type phenotype.Also can implement to use derived from the cell of relevant animal and measure screening based on cell to the influence of cyclic adenosine monophosphate concentration in the born of the same parents.Discovery can produce the candidate compound of this influence and think GPR86 activator or analog.For example, this excitomotor can be used for recovering or improves stimulating, for example the susceptibility of pain.

In preferred embodiments, transgenosis GPR86 animal, particularly GPR86 knock-out animal (fully afunction) demonstrate listed phenotype among the embodiment, preferably measure by listed test wherein.Therefore, GPR86 animal, particularly GPR86 knock-out animal, preferably demonstrate following each or multinomial: the susceptibility of pain is reduced (hypalgesia), the neurological susceptibility of inflammation is reduced.

In highly preferred embodiment, transgenosis GPR86 animal, the particularly measurement parameter of GPR86 knock-out animal demonstrate than the wild-type mice of correspondence high or low (depending on the circumstances) 10%, preferably at least 20%, more preferably at least 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% at least.Therefore, for example, in when test of wagging the tail of listing in an embodiment, the GPR86 knock-out animal has than wild-type mice to be increased by 1 second, 2 seconds, 5 seconds, 10 seconds, 30 seconds or more or 5%, 10%, 20%, 50% or the more threshold of pain that improves.

Obviously, the phenotype of present disclosed GPR86 defective transgenic animals can be used for utilizing the wild type animal, the screening of the compound that the effect that detection causes is similar to the effect of GPR86 afunction.In other words, the wild type animal can be exposed to candidate compound, observe the variation of relevant GPR86 phenotype, such as pain sensitivity reduction etc. to identify the instrumentality, particularly antagonist of GPR86 function.Utilize other local method of identifying of presents, can also detect cell phenotype, such as the variation of cyclic adenosine monophosphate level in the born of the same parents.

Can be used as the antagonist of GPR86 by the compound of this type of Screening and Identification, for example as anodyne, especially for treatment or alleviate the GPR86 relevant disease.

Above-mentioned screening can relate to observes any suitable parameter, such as behavior, physiology or biochemical reaction.Preferred reaction comprises following one or multinomial: the variation of disease resistance; The inflammatory reaction that changes; The tumor susceptibility that changes: the variation of blood pressure; Neovascularization; The variation of feed behavior; The variation of body weight; Changes of bone mineral density; The variation of body temperature; Insulin secretion; Gonadotrophin secretion; Nose and bronchus secretion; Vessel retraction; The loss of memory; Anxiety; Hyporeflexia or exagger; Pain or stress reaction.

Tissue derived from the GPR86 knock-out animal can be used for the receptors bind determination method to determine that whether potential drug (candidate ligand or compound) is in conjunction with the GPR86 acceptor.This type of determination method can be subjected to body preparation by obtaining first from the transgenic animals that are transformed into GPR86 acceptor generation defective, has identified that in conjunction with any the source acquisition second of GPR86 part or compound is implemented by body preparation from known.Usually, first and second are subjected to body preparation except that the source difference of obtaining them, all are similar in all respects.For example, if in determination method, use brain tissue from transgenic animals (as mentioned all and hereinafter described), use suitable brain tissue from normal (wild type) animal so as second source that is subjected to body preparation.Be incubated with known part separately with under the situation that has candidate ligand or compound by body preparation with every kind in conjunction with the GPR86 acceptor.Preferably, check candidate ligand or compound at several variable concentrations.

Be subjected to body preparation to measure the degree of the known ligand combination of replacing to first and second by test compounds.Can in mensuration, directly use tissue, perhaps can handle tissue to separate film or the memebrane protein that itself is used to measure derived from transgenic animals.A kind of preferred transgenic animals are mouse.Can utilize any means compatible to come tagged ligand with binding assay.This will include but not limited to radioactivity, enzyme, fluorescence or chemiluminescent labeling (and above other labelling technique) in greater detail.

And, can use candidate compound etc. by the wild type animal of giving expressive function GPR86, and identify which animal demonstrates and reduce with the expression of GPR86 function of receptors or eliminate the antagonist that relevant any phenotypic characteristic is identified the GPR86 acceptor.

Hereinafter more detailed description be used to generate non-human transgenic animal's detailed method.Genetically modified gene constructs can be imported animal kind system to produce transgene mammal.For example, can the construction of one or several copy be mixed the genome of mammal embryo by the standard transgenic technology.

In the embodiment of a demonstration, generate transgenic nonhuman animal described herein by the kind system that transgenosis is imported the non-human animal.The embryo target cell of each stage of development can be used for importing transgenosis.Can use diverse ways according to the stage of development of embryo target cell.The particular animals strain that is used for this purpose is selected good general health, good embryo production, good embryo Central Plains nuclear visibility and good reproductive fitness.In addition, haplotype is a key factor.

Can realize transgenosis is imported the embryo such as for example microinjection, electroporation or lipofection by any means known in the art.For example, by in the protokaryon of fertilization mammalian ovum, the construction of one or more copies being retained in the mammalian cell of growing the construction microinjection, GPR86 acceptor transgenosis can be imported mammal.After transgenosis construct imported embryonated egg, can be with ovum in different time of external incubation or implant again among the replace-conceive host, perhaps both.External incubation maturation is in the scope of presents.A kind of common method be with the embryo at the about 1-7 of external incubation days (according to species), then they are implanted among the replace-conceive host again.

Southern engram analysis by organizing sections can be to the existence of transgeneic procedure embryo's filial generation test builds thing.If the external source of one or more copies clone construction keeps stable integration in the genome of described transgenic embryo, the permanent transgene mammal that might set up the construction that carries the transgenosis interpolation so is.

Whether can measure construction to the mammiferous young baby that transgenosis changes after the birth is incorporated in the filial generation genome.Preferably, this mensuration is by realizing to the chromosome material from filial generation for the dna sequence dna of expecting the recombinant protein product in encoding or the probe hybridization of its fragment.Discovery comprises those mammal offsprings energy growth and maturity of the construction of at least one copy in its genome.

For the purpose of presents, zygote finger-type in essence becomes to develop into the diploid cell of complete biosome.Usually, zygote will constitute by comprising following nuclear ovum, and described nucleus is natural or artificial formation by merging two haploid cell nuclears from gamete.Therefore, gametid nuclear must be natural compatible nucleus, promptly produces can experience differentiation and grow and become the nucleus of the survived zygote of functional living being body.Usually, preferred euploid zygote.If obtain the aneuploid zygote, chromosome number will be no more than one with respect to the variation of the euploid number of the biosome that therefrom produces gamete so.

Except that similar biology was considered, physics was considered also to determine to add to the zygote nucleus or the amount (for example volume) of giving the exogenous genetic material of the inhereditary material that forms a zygote nucleus part.If do not remove inhereditary material, the amount of so addible exogenous genetic material is subjected to the absorption quantitative limitation of physically not breaking.Usually, the volume of the exogenous genetic material of insertion is no more than about 10 skin liters.To such an extent as to the physical influence of adding should not destroyed the viablity of zygote too greatly physically.The number of dna sequence dna and the restriction of the biology of kind will change with the function of concrete zygote and exogenous genetic material, this is conspicuous for those skilled in the art, because the inhereditary material of gained zygote comprises that exogenous genetic material biologically must start and keep zygote to break up and develop into the functional living being body.

The copy number that adds the transgenosis construct of giving zygote depends on the sum of the exogenous genetic material of interpolation, and will be the quantity that genetic transformation can be taken place.Only need a copy in theory; But, in order to ensure a copy function is arranged, use many copies usually, for example 1,000-20, the transgenosis construct of 000 copy.Advantageously, each exogenous DNA array that inserts has above a functional copy to strengthen the phenotypic expression of exogenous DNA array.

Can use permission to add exogenous genetic material in the nucleus inhereditary material any technology, as long as the cell or the genetic structure of this technology pair cell, nuclear membrane or other existence do not have destructiveness.Preferably exogenous genetic material is inserted the nucleus inhereditary material by microinjection.Cell and cyto-architectural microinjection are the technology that this area has been known and used.

Again implanting utilizes standard method to finish.Usually, anaesthetize the replace-conceive host, and the embryo is inserted fallopian tubal.Embryo's number of implanting in the specific host changes with species, but suitable with the filial generation number of the natural production of these species usually.

Can screen genetically modified existence and/or expression to replace-conceive host's transgenic progeny by any suitable method.Screening usually is the probe of utilization and genetically modified at least a portion complementation, finishes by Southern trace or Northern engram analysis.Utilization can be used as the alternative or addition method that the screening transgene product exists at the Western engram analysis of the antibody of the coded protein of transgenosis.Typically, from afterbody tissue preparation DNA, and by Southern analysis or pcr analysis transgenosis.Perhaps, utilize Southern to analyze or PCR exists and expresses so that the tissue of highest level express transgenic or cell tests are genetically modified thinking, but this analysis can be used any tissue or cell type.

Be used to assess that transgenosis exists substitutes or addition method includes but not limited to suitable biochemical measurement method, such as tissue staining or enzymatic activity, the flow cytometry etc. of enzyme and/or immunoassay, special sign.Blood analysis also can be used for detecting the existence of transgene product in the blood, and the assessment transgenosis is to the influence of various types of haemocytes and other blood constituent level.

By with transgenic animals and suitable spouse's mating, perhaps inseminate by the ovum and/or the sperm in vitro that will from transgenic animals, obtain, can obtain the filial generation of transgenic animals.When the mating carried out with the spouse, the spouse can yes or no transgenosis and/or knock-out animal; When it was transgenic animals, it can comprise identical or different transgenosis, perhaps both.Perhaps, the spouse can be a parental line.When using inseminatio externalis, the fertilization embryo can be implanted the replace-conceive host or at incubated in vitro, perhaps both.Can utilize method mentioned above or other suitable method that genetically modified existence is assessed in the filial generation of using arbitrary method to obtain.

The transgenic animals that produce according to our description will comprise exogenous genetic material.As mentioned above, in certain embodiments, exogenous genetic material will be the dna sequence dna that causes the GPR86 acceptor to produce.In addition, in this embodiment, sequence is attached to the transcriptional control element that allows transgene product preferably to express, for example promoter in particular cell types.

Can also transgenosis be imported the non-human animal with retroviral infection.The non-human embryo that can grow in vitro culture is to the blastocyst stage.During this period, can be with the target (Jaenich, R. (1976) PNAS 73:1260-1264) of blastomere as retroviral infection.Remove the efficient infection (Manipulating the Mouse Embryo, Hogan eds.ColdSpring Harbor Laboratory Press, Cold Spring Harbor, 1986) that oolemma obtains blastomere by the enzyme processing.Be used to import genetically modified virus carrier system and normally carry genetically modified replication defect type retrovirus (Jahner et al. (1985) PNAS 82:6927-6931; Van der Putten et al. (1985) PNAS 82:6148-6152).Can be easily and obtain transfection efficiently (Van der Putten sees above by on the cell monolayer of viral cellulation, cultivating blastomere; Stewart et al. (1987) EMBO J.6:383-388).Perhaps, can implement to infect in the slower stage.Virus or viral cellulation can be injected blastocoele (Jahner et al. (1982) Nature 298:623-628).The great majority person of foundation is inserted (mosaic) for transgenosis, occurs over just in the cell subclass that forms transgenic nonhuman animal because mix.In addition, the person of foundation can comprise the many places retrovirus at genomic diverse location and insert, and they can separate in filial generation usually.In addition, also might transgenosis be imported kind of system's (Jahner et al. (1982) sees above) by second trimester embryo's intrauterine retroviral infection.

Importing genetically modified the 3rd class target cell is embryonic stem cell (ES).From the PIE of in vitro culture, obtain the ES cell and merge (Evans et al. (1981) Nature 292:154-156 with the embryo; Bradley et al. (1984) Nature 309:255-258; Gossler et al. (1986) PNAS 83:9065-9069; And Robertson et al. (1986) Nature 322:445-448).Can efficiently transgenosis be imported the ES cell by the transduction of DNA transfection or retrovirus-mediated method.With this Transformed E S cell is combined with blastocyst from the non-human animal then.After this this ES cell implanted embryo, and the kind system that facilitates the gained chimaeric animals.Referring to summary Jaenisch, R. (1988) Science 240:1468-1474.

We also provide the non-human transgenic animal, and the characteristics of these transgenic animals are to have the GPR86 gene of change, preferably as mentioned above, and as the model of GPR86 function of receptors.The change of gene comprises deletion or the sudden change of other afunction, introduces the foreign gene of the nucleotide sequence with target or random mutation, introduces foreign gene or its combination from another species.Transgenic animals for change can be isozygoty or heterozygosis.Animal and the biologic activity agent that can be used for screening scalable GPR86 function of receptors derived from its cell.Screening technique is to the specificity of the potential therapy of determining pain and inflammatory disorder such as osteoarthritis, rheumatoid arthritis, asthma, IBS and abnormal anti-property and act on particularly useful.This animal can be used as model and is used for studying the effect of GPR86 acceptor in normal brain activity, the heart, spleen and liver function.

Another aspect relates to and has the endogenous GPR86 gene that function is destroyed, but also carries and express the genetically modified transgenic nonhuman animal of the coding allos GPR86 protein GPR86 of another species (promptly from) in its genome.Preferably, animal is a mouse, and allos GPR86 is people GPR86.Chosen animal that GPR86 rebuilds or can be used for identifying in vivo and at the preparation of vitro inhibition people GPR86 derived from the clone of described animal.For example, can exist and lack under the situation of preparation to be measured, apply for animal or clone and induce stimulation that sends signal by people GPR86 and the reaction of measuring animal or clone.According to the reacting phase ratio when lacking preparation, reaction reduces when having preparation, can identify in vivo or at the preparation of vitro inhibition people GPR86.

We also provide the transgenic nonhuman animal (" GPR86 knock-out animal ") of GPR86 defective.This animal is the low animal of expressing or not expressing the GPR86 activity, low express or do not express preferably to be destroyed or to delete by endogenous GPR86 genome sequence cause.Preferably, this animal is not expressed the GPCR activity.More preferably, animal is not expressed the activity of GPR86 shown in SEQ ID NO:3 or the SEQ ID NO:5.Can produce the GPR86 knock-out animal by multiple means known in the art, as hereinafter in greater detail.

Presents also relates to the nucleic acid construct thing that is used for destroying host cell GPR86 gene on function.This nucleic acid construct thing comprises: a) non-homogeneous replacement part; B) be positioned at first homologous region of non-homogeneous replacement part upstream, first homologous region has the nucleotide sequence that essence homogeneity is arranged with a GPR86 gene order; And c) is positioned at second homologous region of non-homogeneous replacement portion downstream, second homologous region has the nucleotide sequence that essence homogeneity is arranged with the 2nd GPR86 gene order, in the endogenous GPR86 gene of natural generation, the 2nd GPR86 gene order is positioned at the downstream of a GPR86 gene order.In addition, first and second homologous regions have enough length to make when nucleic acid molecules imports host cell, between the endogenous GPR86 gene in nucleic acid construct thing and the host cell homologous recombination can take place.In a preferred embodiment, non-homogeneous replacement part comprises the expression reporter, preferably comprises lacZ and is just selecting expression cassette, preferably comprises the neomycin phosphotransferase gene that can be operatively connected with regulating element.

Preferably, the first and second GPR86 gene orders are derived from SEQ ID NO:1, SEQ IDNO:2 or SEQ ID NO:4 or its homolog, variant or derivant.

Another aspect relates to the recombinant vector that has wherein mixed nucleic acid construct thing described herein.Also have an aspect to relate to and wherein imported nucleic acid construct thing described herein, thereby between the endogenous GPR86 gene of permission nucleic acid construct thing and host cell homologous recombination takes place, cause endogenous GPR86 gene that the host cell of functional destruction takes place.Host cell can be from liver, brain, spleen or the heart, and the mammalian cell of normal expression GPR86, or pluripotent cell are such as mouse embryo stem cell.The nucleic acid construct thing has imported wherein and has produced following transgenic nonhuman animal with the further growth of the embryonic stem cell of endogenous GPR86 gene generation homologous recombination, it is filial generation from embryonic stem cell that this animal has, and therefore carries the cell of the GPR86 gene of destruction in its genome.Carry the animal of the GPR86 gene of destruction in can being chosen in its kind then and being, and breed the animal that is created in the GPR86 gene that all has destruction in all body cells and the reproduction cell.This mouse can be bred the GPR86 gene that is paired in destruction then is purifying.

Antibody

For the purpose of presents, unless opposite regulations is arranged, term " antibody " includes but not limited to polyclonal antibody, monoclonal antibody, chimeric antibody, single-chain antibody, Fab fragment and the fragment that produces by the Fab expression library.These fragments comprise and have kept fragment, Fv, F (ab ') and the F (ab ') of complete antibody in conjunction with target material activity ₂Fragment, and single-chain antibody (scFv), fusion and other comprise the synthetic proteins of the antigen binding site of antibody.Antibody and fragment thereof can be humanized antibodies, for example describe among the EP-A-239400.And, also can use to have the antibody of people variable region (or its fragment) completely, for example United States Patent (USP) 5,545, describe in 807 and 6,075,181.Diagnosticum and therapeutic agent especially preferred neutrality antibody, i.e. those antibody of the biologic activity of inhibiting substances amino acid sequence.

Can pass through standard technique, such as immunity inoculation or utilize the phage display storehouse to produce antibody.

Can produce antibody with GPR86 polypeptide or peptide by known technology.This antibody may specificity in conjunction with GPR86 protein or homolog, fragment etc.

If want polyclonal antibody, can be with the selected mammal (for example mouse, rabbit, goat, horse etc.) of the immunogenic composition immunity that comprises GPR86 polypeptide or peptide.According to host species, can use multiple adjuvant to come the enhance immunity reaction.Described adjuvant includes but not limited to Freund, mineral coagulant such as aluminium hydroxide and surface reactive material such as lysolecithin, pluronic polyvalent alcohol, polyanion, peptide, oil emu, key hole  hemocyanin and dinitrophenol dinitrophenolate.If the material amino acid sequence of purifying is administered to the individuality of immunocompromised host for the stimulating system defence, people's adjuvant BCG (Bacille Calmette-Guerin) and the spillikin bacillus (Corynebacterium parvum) that can use potentially useful so.

Collect serum from immune animal, and handle according to known procedures.Comprise antibody if comprise serum at the polyclonal antibody of the epi-position that can obtain from the GPR86 polypeptide, can come the purifying polyclonal antibody by immunoaffinity chromatography at other antigen.The technology that is used to generate and handle polyclonal antiserum is known in this area.In order to generate this antibody, we also provide as haptens and have adhered to (haptenised) in the GPR86 amino acid sequence that is used as immunogenic another amino acid sequence in the animal or human or its fragment.

Those skilled in the art can also be easy to produce the monoclonal antibody at the epi-position that can obtain from GPR86 polypeptide or peptide.The conventional method that produces monoclonal antibody by hybridoma is well-known.Can pass through Fusion of Cells, and other technology, such as transforming bone-marrow-derived lymphocyte with the carcinogenicity dna direct or can producing immortal antibody-producting cell system with the Epstein-Barr virus transfection.Can screen various characteristics to the monoclonal antibody group of respectively organizing that produces at track epi-position (orbitepitope); Be isotype and epi-position affinity.

Can utilize any technology that produces antibody molecule by the continuous cell line of cultivating to prepare monoclonal antibody.These technology include but not limited at first the hybridoma technology described by Koehler and Milstein (1975 Nature 256:495-497); Trioma (trioma) technology, human B cell hybridoma technology (Kosbor et al (1983) Immunol Today 4:72; Cote et al (1983) Proc Natl AcadSci 80:2026-2030) and EBV hybridoma technology (Cote et al., Monoclonal Antibodies andCancer Therapy, pp.77-96, Alan R.Liss, Inc., 1985).

In addition, can use to generating the technology that " chimeric antibody " researched and developed, be about to the splicing of mouse antibodies gene and human immunoglobulin gene to obtain molecule (Morrisonet al (1984) Proc Natl Acad Sci 81:6851-6855 with suitable antigentic specificity and biologic activity; Neuberger et al (1984) Nature312:604-608; Takeda et al (1985) Nature 314:452-454).Perhaps, can change to generating technology (United States Patent (USP) 4,946,779) that single-chain antibody describes to produce the special single-chain antibody of material.

At the antibody of the epi-position that can from GPR86 polypeptide or peptide, obtain, comprise monoclonal and polyclonal, particularly useful in diagnosis, and the antibody of neutrality can be used for passive immunization therapy.Particularly, monoclonal antibody can be used for causing anti-idiotype.Anti-idiotype is to carry the immunoglobulin (Ig) of expecting " inner image (the internal image) " of the material of its protection and/or preparation.Be used to cause that the technology of anti-idiotype is known in this area.These anti-idiotypes also can be used for treatment.

Also can be by inducing generation in the body in lymphocyte populations, perhaps by (1989, Proc Natl Acad Sci 86:3833-3837) and Winter G and Milstein C (1991 such as screening Orlandi; Nature 349:293-299) disclosed recombination immunoglobulin storehouse or high degree of specificity binding reagents group produce antibody.

Also can generate the antibody fragment of the specific binding site that comprises polypeptide or peptide.For example, this fragment includes but not limited to pass through the F (ab ') that the pepsin digested antibody molecule produces ₂Fragment and can by the reduction F (ab ') ₂The Fab fragment that the disulfide bond of fragment produces.Perhaps, can make up the Fab expression library and want specific monoclonal Fab fragment (Huse WD et al (1989) Science 256:1275-1281) to allow to have with easy evaluation rapidly.

Can also change the technology (United States Patent (USP) 4,946,778) that is used to generate single-chain antibody to generate the single-chain antibody of GPR86 polypeptide.Equally, transgenic mice or other biosome comprise that other mammal also can be used for expressing humanized antibody.

Above-mentioned antibody can be used for separating or identifies the clone of express polypeptide or be used for coming purified polypeptide by affinity chromatography.

Antibody at the GPR86 polypeptide also can be used for treating pain and inflammatory disorder, such as osteoarthritis, rheumatoid arthritis, asthma, IBS and allergic reaction.

The diagnostic assay method

We also described GPR86 polynucleotide and polypeptide (and homolog, variant and derivant) in diagnosis as diagnosticum or the purposes in genetic analysis.With complementary nucleic acid of GPR86 nucleic acid (comprising homolog, variant and derivant) or the nucleic acid that can hybridize with it, and also be useful in this type of determination method at the antibody of GPR86 polypeptide.

The mutant form that detects the GPR86 gene relevant with dysfunction will provide the instrument that can add in following disease or the disease susceptibility diagnosis or limit following disease or disease susceptibility diagnosis, and described disease is to be changed by the expression deficiency of GPR86, over-expression or expression to cause.Can detect the individuality that carries sudden change in the GPR86 gene (comprising control sequence) at dna level by multiple technologies.

For example, can be from the patient DNA isolation, and measure the dna polymorphism pattern of GPR86.The pattern that evaluation draws is compared with the control patients of the known GPR86 of suffering from over-expression, expression deficiency or abnormal expression relevant disease.Can identify the patient who expresses the genetic polymorphism pattern relevant then with the GPR86 relevant disease.Can implement the genetic analysis of GPR86 gene by any technology known in the art.For example, can be by measuring the allelic dna sequence dna of GPR86, waiting by RFLP or snp analysis and screen individuality.Can identify the patient who GPR86 over-expression, expression deficiency or abnormal expression diseases related is had the genetic predisposition by the existence of the dna polymorphism in the sequence that detects GPR86 gene order or any its expression of control.

The patient that can treat such evaluation is then taken place with prevention GPR86 relevant disease, or the GPR86 relevant disease has more aggressiveness in early days with prophylactic further generation or development.The GPR86 relevant disease comprises the dopamine relevant disease, such as Parkinson's disease, heart disease such as supraventricular or ventricular arrhythmia, low blood pressure, feel sick, tourette syndrome, stress and pain.

We also disclose the kit of the genetic polymorphism pattern relevant with the GPR86 relevant disease that is used to identify the patient.This kit comprises the DNA sampling means and is used to measure the means of genetic polymorphism pattern, can compare with control sample then to determine the neurological susceptibility of patient to the GPR86 relevant disease.The kit that is used to diagnose the GPR86 relevant disease also is provided, has comprised the GPR86 polypeptide and/or at the antibody of this type of polypeptide (or its fragment).

Can be from experimenter's cell, such as obtaining diagnostic nucleic acid in blood, urine, saliva, biopsy or the postmortem material.In a preferred embodiment, DNA obtains from prick the haemocyte that is collected in the blood acquisition on the thieving paper when patient points.In another preferred embodiment, at AmpliCard ^TM(University of Sheffield, Department of Medicine andPharmacology, Royal Hallamshire Hospital, Sheffield, England S10 2JF) goes up and collects blood.

DNA can be directly used in detection, perhaps can utilize PCR or other amplification technique to carry out enzymatic amplification before analysis.In order in the PCR reaction, to realize the amplification of target sequence, can prepare the oligonucleotide DNA primer in specific polymorphic dna district in the target genes of interest.RNA or cDNA also can be used as template in a similar manner.Can utilize dna sequence dna the genetic polymorphism to determine extension increasing sequence exist of restriction enzyme analysis then, thereby patient's genetic polymorphism collection of illustrative plates is provided from the template DNA amplification.Can identify the length of restriction fragment by gel analysis.Perhaps/in addition, can adopt such as technology such as SNP (single nucleotide polymorphism) analyses.

Can detect deletion and insertion by the variation of comparing the amplified production size with normal genotype.Can by will the amplification DNA with identify point mutation through mark GPR86 nucleotide sequence hybridization.Can complete matching sequence and mispairing duplex be differentiated by RNA enzymic digestion or the difference by melting temperature.Also can detect dna sequence dna difference by the change of dna fragmentation electrophoretic mobility in the gel that is with or without denaturant or by direct dna sequencing.Referring to for example Myers et al, Science (1985) 230:1242.Also can protect determination method, disclose the sequence variation of ad-hoc location such as RNA enzyme and S1 protection or chemical cleavage method by nuclease.Referring to Cotton et al., Proc Natl Acad Sci USA (1985) 85:4397-4401.In another embodiment, can make up comprise GPR86 nucleotide sequence or its fragment oligonucleotide probe array to implement efficient screening, for example efficient screening of genetic mutation.The array technique method is well known, and has extensive applicability, can be used for studying the various problems in the molecular genetics, comprise that gene expression, genetic linkage and hereditary variability are (referring to for example M.Chee etal., Science, Vol 274, pp 610-613 (1996)).

Single-strand conformation polymorphism (SSCP) can be used for detecting difference (Orita et al. (1989) Proc.Natl.Acad.Sci.USA:86:2766 of electrophoretic mobility between mutant and the wild-type nucleic acid; Also can be) referring to Cotton (1993) Mutat Res 285:125-144 and Hayashi (1992) Genet Anal Tech Appl 9:73-79.Can make the single stranded DNA fragment generation sex change of sample and contrast GPR86 nucleic acid, and allow their renaturation.The secondary structure of single-chain nucleic acid changes with sequence, and the change of the electrophoretic mobility of generation makes us even can detect the variation of single base.Can use through label probe mark or detection dna fragmentation.Can strengthen the sensitivity of determination method by using the secondary structure RNA (rather than DNA) more responsive to sequence variation.In a preferred embodiment, experimental technique uses the heteroduplex analysis to separate double-stranded heteroduplex molecule (Keen et al (1991) Trends Genet 7:5) with the basis that is changed to of electrophoretic mobility.

The diagnostic assay method provides by described method and has detected sudden change in the GPR86 gene with diagnosis or determine method to susceptibility infection.

But the existence of GPR86 polypeptide and nucleic acid in the test sample.Therefore, can diagnose above listed infection and disease by comprising the method that the horizontal abnormality derived from experimenter's sample determination GPR86 polypeptide or GPR86 mRNA is reduced or improves.Sample can comprise from suffering from or suspecting to suffer from GPR86 and express to improve, reduce or other is unusual, comprise that the space of expression or pattern or time change the cell or tissue sample of the biosome of diseases associated.To suffer from or suspect that it is useful that expression in the expression of GPR86 in the biosome that suffers from this type of disease or pattern and the normal biosome or pattern are compared as the means that diagnose the illness.

Therefore, generally speaking, we disclose by the existence that makes sample and at least a nucleic acid probe to described nucleic acid specificity contact and monitor described sample amplifying nucleic acid and have come to comprise in the test sample method that the nucleic acid of GPR86 nucleic acid exists.For example, but the nucleic acid probe specificity and detects combination between the two in conjunction with GPR86 nucleic acid or its part; Also can detect the existence of compound itself.And we disclose by making cell sample contact detect the method that the GPR86 polypeptide exists in conjunction with the antibody of polypeptide and the existence of monitoring polypeptide in the described sample.This can realize easily by existence of monitoring the compound that forms between antibody and the polypeptide or the combination of monitoring between polypeptide and the antibody.The method that detects combination between two entities is known in this area, comprises FRET (FRET (fluorescence resonance energy transfer)), surperficial excimer resonance etc.

Can utilize any method of quantification polynucleotide well-known in the art, measure the expression that reduces or improve at rna level such as for example PCR, RT-PCR, RNA enzyme protection, Northern trace and other hybridizing method.Can be used for measuring protein level, know for those skilled in the art such as the determination techniques of GPR86 level derived from host's the sample.This type of assay method comprises radioimmunoassay, competitive binding assay method, Western engram analysis and ELISA determination method.

This open diagnostic kit that also relates to disease or disease susceptibility (comprising infection), described disease is the dopamine relevant disease for example, such as Parkinson's disease, heart disease such as supraventricular or ventricular arrhythmia, low blood pressure, feel sick, tourette syndrome, stress and pain.Diagnostic kit comprises GPR86 polynucleotide or its fragment; Complementary nucleotide sequence; GPR86 polypeptide or its fragment, or at the antibody of GPR86 polypeptide.

The chromosome determination method

The GPR86 nucleotide sequence identifies it also is valuable to chromosome.Specific site on the individual human chromosome of this sequence specific target also can be hybridized with it.As mentioned above, finder GPR86 is positioned at human chromosomal 3q24.

The mapping of chromosome correlated series is the important first step with those sequences and gene-correlation disease association.In case sequence is positioned accurate chromosome position, so just the physical location of this sequence on the chromosome can be associated with the genetic map data.For example, at V.McKusic k, can find this data among the Mendelian heritancein Man (can by the online acquisition of Johns Hopkins University Welch Medical Library).Then by linkage analysis (the common heredity of adjacent gene physically) identified gene be positioned relation between the disease of identical chromosomal region.

Also can determine the difference of cDNA between the influenced and impregnable individuality or genome sequence.If observe sudden change in some or whole affected individuality, but do not observe this sudden change in any normal individual, this sudden change is likely the paathogenic factor of disease so.

Prevention and methods of treatment

We also provide the method for the excessive and not enough relevant unusual condition of treatment and GPR86 activity.

If the GPR86 activity is excessive, can utilize several method.A kind of method comprises the inhibitor compound mentioned above (antagonist) of using effective dose to the experimenter with pharmaceutically acceptable carrier suppressing activation by block ligand in conjunction with GPR86 or by suppressing secondary signal, thereby alleviates unusual condition.

In another approach, can use the soluble form that still can compete the GPR86 polypeptide of binding partner with endogenous GPR86.The representative instance of this competition thing comprises the fragment of GPR86 polypeptide.

In also having a kind of method, can utilize and express interrupter technique suppress the to encode expression of gene of endogenous GPR86.Known this technology relates to utilizes inner antisense sequences that produce or separate administration.Referring to for example O ' Connor, J Neurochem (1991) 56:560 in Oligodeoxvnucleotides asAntisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla. (1988).Perhaps, can provide the oligonucleotides that forms triple helix with gene.Referring to for example Lee et al., NucleicAcids Res (1979) 6:3073; Cooney et al., Science (1988) 241:456; Dervan et al., Science (1991) 251:1360.These oligomers itself can be used, perhaps relevant oligomer can be expressed in vivo.

To express and active relevant unusual condition in order treating, also can to utilize several method with GPR86 is low.A kind of method comprises with pharmaceutically acceptable carrier unites compound to the activation GPR86 of experimenter's administering therapeutic effective dose, activator promptly mentioned above, thus alleviate unusual condition.Perhaps, can use gene therapy to influence the GPR86 endogenous production of relevant cell among the experimenter.For example, in order in aforesaid replication defect type retroviral vector, to express, can transform the GPR86 polynucleotide.Separable then retrovirus expression construction, and with in the incasing cells of its importing with the retroviral plasmid vector transduction of the RNA that comprises coding GPR86 polypeptide, incasing cells just produces the infectious viral particle that comprises genes of interest now like this.These producer's cells the experimenter be can be administered to, engineered cells and express polypeptide in vivo in vivo are used for.The summary of gene therapy is referring to Chapter 20, Gene Therapyand other Molecular Genetic-based Therapeutic Approaches (and list of references of quoting) in Human Molecular Genetics, T Strachan and A P Read, BIOS ScientificPublishers Ltd (1996).

Prepare and use

Can unite with suitable pharmaceutical carrier and prepare peptide, such as soluble form and activator and the antagonist peptide or the micromolecule of GPR86 polypeptide.This type of preparaton comprises polypeptide or compound and the pharmaceutically acceptable carrier or the excipient for the treatment of effective dose.Described carrier includes but not limited to salt solution, buffer saline, dextrose, water, glycerine, ethanol and combination thereof.Preparaton should be suitable for mode of administration, and this is fully in the technical scope of this area.Presents also relates to pharmaceutical pack and the kit that comprises one or more one or more components of filling above-mentioned composition.

GPR86 polypeptide and other compound can use separately or with other compound, unite use such as therapeutic compound.

The preferred form of the systemic application of Pharmaceutical composition comprises injection, typically by intravenous injection.Can use other injection path, such as subcutaneous, intramuscular or intraperitoneal.The alternative of system's dispenser comprise utilize bleeding agent such as cholate or fusidinic acid or other detergent through mucous membrane with through the skin dispenser.In addition, if suitably be mixed with enteron aisle or dress capsule preparaton, so oral dispenser also is possible.Can also paste, forms such as paste, gel are local and/or these compounds are used in the location.

The dosage range that needs depends on peptide, dispenser path, the character of preparaton, the character of experimenter's situation and the doctor in charge's the judgement of selection.But suitable dosage is within 0.1-100 μ g/kg experimenter's scope.But because the diversity of available compound and the different efficient in various dispensers path, the dosage that needs exists the variation of broad also in expectation.For example, the dosage that oral dispenser expection need be higher than intravenous injection dispenser.The standard normal experience optimization that utilizes this area to understand fully can be adjusted the variation of these dosage levels.

Polypeptide that also can endogenous generation is used for the treatment of in the experimenter usually is called " gene therapy ", as mentioned above on the treatment form.Therefore, for example, can use the polynucleotide of ex vivo (ex vivo) coded polypeptide,, transform cell, for example by utilizing retroviral plasmid vector from the experimenter such as DNA or RNA.Then cell is imported the experimenter.

Pharmaceutical composition

We also provide the Pharmaceutical composition of the GPR86 polypeptide described herein, polynucleotide, peptide, carrier or its antibody that comprise the administering therapeutic effective dose and optional pharmaceutically acceptable carrier, thinning agent or excipient (comprising its combination).

Pharmaceutical composition can use for the human or animal in people and veterinary science, and typically comprises any or multiple pharmacy acceptable diluent, carrier or excipient.The acceptable carrier or the thinning agent that use for treatment are well known at drug world, and at for example Remington ' s PharmaceuticalSciences, among the Mack Publishing Co. (A.R.Gennaro edit.1985) description are arranged.Can put into practice according to the pharmacy of predetermined dispenser path and standard and select pharmaceutical carrier, excipient or thinning agent.Pharmaceutical composition can comprise any suitable adhesive, lubricant, suspending agent, dressing material, the solubilizer as carrier, excipient or thinning agent or comprise any suitable adhesive, lubricant, suspending agent, dressing material, solubilizer outside carrier, excipient or thinning agent.

Antiseptic, stabilizing agent, dyestuff even flavoring additives can be provided in Pharmaceutical composition.The example of antiseptic comprises the ester of Sodium Benzoate, sorbic acid and P-hydroxybenzoic acid.Also can use antioxidant and suspending agent.

Different compositions/formulation requirement can be arranged, and this depends on different delivery systems.For instance, Pharmaceutical composition described herein can be mixed with the form of utilizing micropump or sending by the mucous membrane path, nasal spray or for example for the aerosol that sucks or absorbable solution or the form by parenteral delivery, wherein composition is mixed with injectable form, for example passes through the form of intravenous, intramuscular or the administration of subcutaneous path.Perhaps, preparaton can be designed to can be by these two kinds of forms that approach is sent.

When by gastrointestinal mucosa transmucosal delivery medicament, medicament should be able to be stable by maintenance during the intestines and stomach; For example, it should have resistibility to proteoclastic degraded, keeps stable at acid pH, and the decontamination effect of bile is had resistibility.

If suitably, can by suck, with the form of suppository or pessary, with the form of local lotion, solution, creme, ointment or face powder, use dermal patch, with the tablet that contains excipient such as starch or lactose, separately or with the capsule of mixed with excipients or capsule and pill (ovule) form is oral or use Pharmaceutical composition with the elixir, solution or the form of suspension that contain flavoring additives or colorant, perhaps can inject outward by stomach and intestine, for example intravenous, intramuscular or subcutaneous.In order to carry out the parenteral dispenser,, for example make the form of the aseptic aqueous solution of capacity salt that solution and blood etc. ooze or monose use composition preferably can comprise other material.In order to carry out oral cavity or hypogloeeis dispenser, tablet or the lozenge form that can prepare are in a usual manner used composition.

Vaccine

Another embodiment relates to the method that is used in the reaction of mammal induction of immunity; this method comprises to the enough GPR86 polypeptide of mammal inoculation or its fragment protects described animal to avoid the GPR86 relevant disease to produce antibody and/or t cell immune response.

Another embodiment relates to the method for induction of immunity reaction in mammal, and this method comprises with the carrier that instructs the GPR86 polynucleotide to express in vivo sends the GPR86 polypeptide to induce described immune response, produces antibody and protects described animal to avoid disease.

When another embodiment relates in importing mammalian hosts, induce the immunoreactive immunity/vaccine mixture (composition) at the GPR86 polypeptide in this mammal, wherein composition comprises GPR86 polypeptide or GPR86 gene.Vaccine mixture also can comprise appropriate carriers.

Therefore because the GPR86 polypeptide may be decomposed under one's belt, preferably use (comprise in subcutaneous, intramuscular, intravenous, the corium etc. injection) by the stomach and intestine outer pathway.The formulation that is suitable for the parenteral dispenser comprises moisture and anhydrous aseptic injectable solution, and they can contain antioxidant, buffering agent, bacteriostatic agent and make preparaton and solute that recipient's blood etc. oozes; And moisture and anhydrous sterile suspensions, but their suspending agent-containings or thickening agent.Preparaton may reside in unit dose or the multi-dose container, for example in Mi Feng ampoule and the phial, and can be kept in the freeze-dried condition, only needs add immediately before use sterile liquid carrier.Vaccine mixture also can comprise the immunogenic adjuvant system that is used to strengthen preparaton, for example oil-in-water system and other system known in the art.Dosage will depend on that the ratio of vaccine is alive, and can determine easily by normal experiment.

Can prepare vaccine from one or more GPR86 polypeptide or peptide.

It is known for those skilled in the art as the bacterin preparation of active component to comprise immunogenic polypeptide or peptide.Typically, this vaccine production can be become injectable forms, for example liquid solution or suspending liquid; Also can prepare and be suitable for before injection dissolving or be suspended in solid form in the liquid.Also but emulsification preparation perhaps is encapsulated in protein in the liposome.Usually with active immne originality composition and pharmacy can be accepted and the mixed with excipients compatible with active component.Appropriate excipients is for example water, salt solution, dextrose, glycerine, ethanol etc. and combination thereof.

In addition, if desired, vaccine can comprise a spot of auxiliary substance, such as the adjuvant of wetting agent or emulsifying agent, pH buffering agent and/or enhancing vaccine potency.Effectively the example of adjuvant includes but not limited to aluminium hydroxide, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-just-muramyl-(CGP 11637 for L-alanyl-D-isoglutamine, just-MDP) be called, the different glutamy of N-acetyl muramyl-L-alanyl-D--L-alanine-2-(1 '-2 '-two palmityls-sn-glycerine-3-hydroxyl phosphinylidyne oxygen)-ethamine (CGP 19835A, be called MTP-PE), and RIBI, it comprises three kinds of compositions that extract, monophosphoryl lipid A from bacterium in 2% squalene/Tween 80 emulsion fluid, two mycolic acid trehaloses and cell wall skeleton (MPL+TDM+CWS).

More examples of adjuvant and other preparation comprise aluminium hydroxide, aluminum phosphate, aluminium potassium sulfate (alum), beryllium sulfate, tripoli, porcelain earth, carbon, water-in-oil emulsion, oil-water emulsifiers, muramyl dipeptide, bacterial endotoxin, lipid X, spillikin bacillus (Propionibacterium (Propionobacterium acnes)), Bordetella pertussis (Bordetella pertussis), polyribonucleotide, sodium alginate, sheep oil, lysolecithin, vitamin A, saponin, liposome, levamisol, the DEAE-dextran, segmented copolymer or other synthetic adjuvant.These adjuvants can be bought from various sources, for example the Merck adjuvant 65 (Merck and Company, Inc., Rahway, N.J.) or incomplete Freund and Freund's complete adjuvant (Difco Laboratories, Detroit, Michigan).

Typically, the adjuvants such as potpourri of use such as Amphigen (oil-in-water), Alhydrogel (aluminium hydroxide) or Amphigen and Alhydrogel.Have only the aluminium hydroxide approval to use for the people.

The ratio of immunogene and adjuvant can change in the scope of broad, as long as the both exists with effective dose.For example, can there be (Al in aluminium hydroxide with about 0.5% amount of vaccine mixture ₂O ₃The basis).Be easily, vaccine is mixed with and comprises the immunogene of final concentration in 0.2-200 μ g/ml scope, preferred 5-50 μ g/ml, most preferably 15 μ g/ml.

After the preparation, the sterile chamber of vaccine can being packed into seals and is stored in low temperature then, and for example 4 ℃, perhaps can be with its freeze-dried.Freeze-drying allows to store with stable form for a long time.

Conventional by injection, for example subcutaneous or intramuscular, stomach and intestine are used vaccine outward.Other formulation that is suitable for other dispenser pattern comprises the peroral dosage form in suppository and some situation.For suppository, conventional adhesive and carrier can comprise for example polyolefin glycol or triglyceride; Can be by containing 0.5%-10%, the potpourri of the active component of preferred 1%-2% forms this suppository.Peroral dosage form comprises the excipient of normal use, such as the sweet mellow wine of for example pharmaceutical grade, lactose, starch, dolomol, saccharin sodium, cellulose, magnesium carbonate etc.These compositions can be taked solution, suspending liquid, tablet, pill, capsule, extended release preparation or powder form, and contain the active component of 10%-95%, preferred 25%-70%.During with the vaccine combination freeze-drying, can before dispenser, the freeze-drying raw material be reconstructed into for example suspending liquid.Preferably in damping fluid, finish reconstruction.

Can the casing of Eudragit (acryl resin) " S ", Eudragit " L ", cellulose acetate, phthalandione cellulose acetate or hydroxypropyl methylcellulose be provided for to the oral capsule of patient, tablet and pill with for example comprising.

Can the GPR86 polypeptide be formulated in the vaccine with neutral form or salt form.The acceptable salt of pharmacy comprises and mineral acid, such as for example hydrochloric acid or phosphoric acid, and the perhaps acid-addition salts (forming) that forms of organic acid such as acetic acid, oxalic acid, tartrate and maleic acid with the free amine group of peptide.The salt that forms with free carboxy also can be derived from inorganic base, such as for example NaOH, potassium, ammonium, calcium or iron, and organic base, such as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine and procaine.

Dispenser

Typically, the internist will determine to be suitable for most the actual dose of experimenter's individuality, and it will change with age, body weight and the reaction of particular patient.Following dosage is the illustration of average case.Certainly, can have need be higher or than the individual instances of low dosage scope.

Can use Pharmaceutical composition described herein and vaccine combination by direct injection.Composition can be mixed with and be used for parenteral, mucous membrane, intramuscular, intravenous, subcutaneous, intraocular or through the skin dispenser.Typically, can be with the 0.01-30mg/kg body weight, preferred 0.1-10mg/kg body weight, more preferably the dosage of 0.1-lmg/kg body weight is used every kind of protein.

Term administering " comprise by virus or the sending of non-virus technology.The virus delivery mechanism includes but not limited to adenovirus vector, adeno-associated virus (AAV) carrier, herpesvirus vector, retroviral vector, slow virus carrier and baculovirus vector.Non-viral delivery mechanism comprises transfection, liposome, immunoliposome, lipofectin (lipofectin reagent), cationic surface amphipath (CFA) and the combination thereof of lipid mediation.The path of these delivery mechanisms includes but not limited to mucous membrane, nose, oral, parenteral, stomach and intestine, part or path, hypogloeeis.

Term administering " include but not limited to send nasal spray or for example for the aerosol or the absorbable solution that suck by the mucous membrane path; Send by the parenteral path, wherein by injectable form, for example send in intravenous, intramuscular or subcutaneous path.

Term " is used altogether " and is referred to that for example GPR86 polypeptide and additional entities such as dispenser position and the time of each in the adjuvant can be realized immune necessary the adjusting.Therefore, although can use polypeptide and adjuvant with same position at one time, it may be favourable using polypeptide at the time different with adjuvant and different positions.Even can in identical delivery medium, send polypeptide and adjuvant, and polypeptide and antigen can be coupling and/or not coupling and/or in heredity coupling and/or not coupling.

Can be with potion or multi-agent with GPR86 polypeptide, polynucleotide, peptide, nucleotide or its antibody and optional adjuvant separate administration or be administered to experimenter host altogether.

Can pass through many different paths, such as in injection (comprising parenteral, subcutaneous and intramuscular injection), the nose, in the mucous membrane, oral, vagina, urethra or eye dispenser use vaccine combination described herein and Pharmaceutical composition.

Can be by injection, for example subcutaneous or intramuscular injection, the outer routine of stomach and intestine is used vaccine described herein and Pharmaceutical composition.Other formulation that is suitable for other dispenser pattern comprises the peroral dosage form in suppository and some situation.For suppository, conventional adhesive and carrier can comprise for example polyolefin glycol or triglyceride; Can be that the potpourri of the active component of 1%-2% forms this suppository by containing 0.5%-10%.Peroral dosage form comprises the excipient of normal use, such as the sweet mellow wine of for example pharmaceutical grade, lactose, starch, dolomol, saccharin sodium, cellulose, magnesium carbonate etc.These compositions can be taked solution, suspending liquid, tablet, pill, capsule, sustained release forms or powder form, and contain the active component of 10%-95%, preferred 25%-70%.During with the vaccine combination freeze-drying, can before dispenser, the freeze-drying raw material be reconstructed into for example suspending liquid.Preferably in damping fluid, finish reconstruction.

Aspect further

The present invention further aspect and embodiment have been listed hereinafter in Bian Hao the paragraph; Should understand the present invention and contain these aspects:

Paragraph 1: comprise the GPR86 polypeptide of amino acid sequence shown in SEQ ID NO:3 or the SEQ ID NO:5, or its homolog, variant or derivant.

Paragraph 2: coding is according to the nucleic acid of the polypeptide of paragraph 1.

Paragraph 3: according to the nucleic acid of paragraph 2, comprise nucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:2 or the SEQID NO:4, or its homolog, variant or derivant.

Paragraph 4: comprise polypeptide according to the fragment of the polypeptide of paragraph 1.

Paragraph 5: according to the polypeptide of paragraph 3, comprise one or more zones of homology between SEQ ID NO:3 and the SEQ ID NO:5, perhaps comprise one or more zones of allos between SEQ ID NO:3 and the SEQ ID NO:5.

Paragraph 6: coding is according to the nucleic acid of the polypeptide of paragraph 4 or 5.

Paragraph 7: comprise carrier according to the nucleic acid of paragraph 2,3 or 6.

Paragraph 8: comprise according to the nucleic acid of paragraph 2,3 or 6 or according to the host cell of the carrier of paragraph 7.

Paragraph 9: comprise according to the nucleic acid of paragraph 2,3 or 6 or according to the transgenic nonhuman animal of the carrier of paragraph 7.

Paragraph 10:, be mouse according to the transgenic nonhuman animal of paragraph 9.

Paragraph 11: according to the polypeptide of paragraph 1,4 or 5 identify can with the purposes in the method for the interactional compound of g protein coupled receptor specificity.

Paragraph 12: according to the transgenic nonhuman animal of paragraph 9 or 10 identify can with the purposes in the method for the interactional compound of g protein coupled receptor specificity.

Paragraph 13: be used for identifying the method for the compound that can improve cell cyclic adenosine monophosphate endogenous levels, whether this method comprises makes cells contacting candidate compound of expressing GPR86 and the level of determining cyclic adenosine monophosphate (cAMP) in the cell owing to described contact improves.

Paragraph 14: be used for identifying the method for the compound that can reduce cell cyclic adenosine monophosphate endogenous levels, whether this method comprises makes cells contacting candidate compound of expressing GPR86 and the level of determining cyclic adenosine monophosphate (cAMP) in the cell owing to described contact reduces.

Paragraph 15: being used to identify can be in conjunction with the method for the compound of GPR86 polypeptide, and this method comprises to be made GPR86 polypeptide contact candidate compound and determine that whether this candidate compound is in conjunction with the GPR86 polypeptide.

Paragraph 16: by according to the arbitrary method compounds identified of paragraph 11-15.

Paragraph 17: can specificity in conjunction with compound according to the polypeptide of paragraph 1,4 or 5.

Paragraph 18: according to polypeptide or its part of paragraph 1,4 or 5, perhaps according to the nucleic acid of paragraph 2,3 or 6 purposes in the method that is used for generating antibody.

Paragraph 19: can specificity in conjunction with according to the polypeptide of paragraph 1,4 or 5 or its part or by according to the polypeptide of the nucleotide coding of paragraph 2,3 or 6 or the antibody of its part.

Paragraph 20: comprise following each or multinomial Pharmaceutical composition: according to polypeptide or its part of paragraph 1,4 or 5; Nucleic acid or its part according to paragraph 2,3 or 6; Carrier according to paragraph 7; Cell according to paragraph 8; Compound according to paragraph 16 or 17; Reach antibody according to paragraph 19, and pharmaceutically acceptable carrier or thinning agent.

Paragraph 21: comprise following each or multinomial vaccine combination: according to polypeptide or its part of paragraph 1,4 or 5; Nucleic acid or its part according to paragraph 2,3 or 6; Carrier according to paragraph 7; Cell according to paragraph 8; Compound according to paragraph 16 or 17; Reach antibody according to paragraph 19.

Paragraph 22: comprise following each or the multinomial disease or the diagnostic kit of disease susceptibility: according to polypeptide or its part of paragraph 1,4 or 5; Nucleic acid or its part according to paragraph 2,3 or 6; Carrier according to paragraph 7; Cell according to paragraph 8; Compound according to paragraph 16 or 17; Reach antibody according to paragraph 19.

Paragraph 23: treatment suffers from the method with the patient of GPR86 increased activity diseases associated, and this method comprises to the patient uses the GPR86 antagonist.

Paragraph 24: treatment suffers from the active method that reduces the patient of diseases associated with GPR86, and this method comprises to the patient uses the GPR86 activator.

Paragraph 25: according to the method for paragraph 23 or 24, wherein GPR86 comprises and has polypeptide of sequence shown in SEQ ID NO:3 or the SEQ ID NO:5.

Paragraph 26: be used for treating and/or preventing the method for disease, comprise to the patient and use following each or multinomial step: according to polypeptide or its part of paragraph 1,4 or 5 the patient; Nucleic acid or its part according to paragraph 2,3 or 6; Carrier according to paragraph 7; Cell according to paragraph 8; Compound according to paragraph 16 or 17; Antibody according to paragraph 19; Pharmaceutical composition according to paragraph 20; Reach vaccine according to paragraph 21.

Paragraph 27: comprise polypeptide or its part according to paragraph 1,4 or 5; Nucleic acid or its part according to paragraph 2,3 or 6; Carrier according to paragraph 7; Cell according to paragraph 8; Compound according to paragraph 16 or 17; And/or according to the medicament of the antibody of paragraph 19, described medicament uses in treatment or prophylactic method.

Paragraph 28: according to polypeptide or its part of paragraph 1,4 or 5; Nucleic acid or its part according to paragraph 2,3 or 6; Carrier according to paragraph 7; Cell according to paragraph 8; Compound according to paragraph 16 or 17; And/or the Antibody Preparation of paragraph 19 is used for the treatment of or the purposes of prophylactic Pharmaceutical composition.

Paragraph 29: the non-human transgenic animal is characterized in that transgenic animals comprise the GPR86 gene of change.

Paragraph 30: according to the non-human transgenic animal of paragraph 29, wherein said change be selected among GPR86 deletion, the GPR86 cause the sudden change of afunction, in GPR86, introduce the foreign gene of nucleotide sequence, in GRP86, introduce foreign gene from another species with target sudden change or random mutation, and above-mentioned every combination in any.

Paragraph 31: the transgenic nonhuman animal that the function of endogenous GPR86 gene is destroyed, wherein said transgenic animals comprise the transgenosis of coding allos GPR86 protein and express this transgenosis in its genome.

Paragraph 32: be used for destroying the nucleic acid construct thing of host cell GPR86 gene function, this nucleic acid construct thing comprises: a) non-homogeneous replacement part; B) be positioned at first homologous region of non-homogeneous replacement part upstream, this first homologous region has the nucleotide sequence that essence homogeneity is arranged with a GPR86 gene order; And c) is positioned at second homologous region of non-homogeneous replacement portion downstream, this second homologous region has the nucleotide sequence that essence homogeneity is arranged with the 2nd GPR86 gene order, and the 2nd GPR86 gene order is positioned at the downstream of a GPR86 gene order in the endogenous GPR86 gene of natural generation.

Paragraph 33: be used to produce the method for GPR86 polypeptide, this method is included in the host cell of cultivating under the condition that the nucleic acid of coding GPR86 polypeptide expresses according to paragraph 8.

Paragraph 34: whether have method in the test sample according to the nucleic acid of paragraph 2,3 or 6, this method comprise make sample contact at least a to described nucleic acid specificity nucleic acid probe and monitor whether there is described nucleic acid in the described sample.

Paragraph 35: whether have the method according to the polypeptide of paragraph 1,4 or 5 in the test sample, this method comprises makes sample contact according to the antibody of paragraph 19 and monitor whether there is described polypeptide in the described sample.

Paragraph 36: diagnosis is expressed by GPR86 and is improved, reduces or other disease that causes unusually or syndrome, perhaps express with GPR86 and improve, reduce or unusual diseases associated or syndromic method, the method comprising the steps of: (a) in expression or the pattern of suffering from or suspect detection GPR86 in the animal that suffers from this type of disease; And (b) expression or pattern and intact animal are compared.

Paragraph 37: according to the kit of paragraph 22, according to the arbitrary method of paragraph 23-26, medicament according to paragraph 27, purposes according to paragraph 28, perhaps according to the method for paragraph 36, wherein said disease is relevant with the inflammatory disorder, is preferably selected from inflammatory disease (rheumatoid arthritis for example, multiple sclerosis, guillain-Barre syndrome, Crohn's disease, ulcerative colitis, psoriasis, graft versus host disease(GVH disease), systemic lupus, lupus erythematosus or insulin-dependent diabetes), autoimmune disease (TSS for example, osteoarthritis, diabetes or inflammatory bowel disease), acute pain, chronic pain, neuropathic pain, contact dermatitis, atherosclerotic, glomerulonephritis, reperfusion injury, bone-resorbing disease, asthma, apoplexy, miocardial infarction, thermal burn, adult respiratory distress syndrome (ARDS) (ARDS), the multiple organ injury of wound secondary, skin disease with the acute inflammation composition, acute purulent meningitis, gangrenosum acne intestines colitis, with haemodialysis, infectious shock, the leucocyte removal art, the syndrome that the granulocyte blood transfusion is relevant, suck the acute or chronic inflammation of lung that causes by smog, mullerianosis, Behcet, uveitis, ankylosing spondylitis, pancreatitis, cancer, Lyme disease, ISR after skin is worn the chamber coronary angioplasty, Alzheimer's, traumatic arthritis, pyemia, chronic obstructive pulmonary disease, congestive heart failure, osteoporosis, cachexia, Parkinson's disease, periodontosis, gout, allergic disease, the age related macular degeneration, infect and cystic fibrosis.

Embodiment

Embodiment 1: transgenosis GPR86 knock-out mice: the structure of GPR86 gene targeting carrier

Utilization has been identified the PAC that comprises the GPR86 gene derived from the radiolabeled probe of coded sequence section from the PAC library.Utilize the internal limitations site anchor PCR similar (in-house restriction site anchored PCR) method to assemble 8.0kb genome contig to GeneWalker (Clontech).Further biological information work is increased to 300kb with the contig size.This contig provides sufficient flanking sequence information, thereby can design the homology arm of being cloned in the target-seeking carrier.

Mouse GPR86 gene has 1 coding extron.Design target strategy is removed most of coded sequence, comprises whole membrane-spanning domain.Wait to delete the 3.9kb 5 ' homology arm and the 1.2kb 3 ' homology arm of district's flank by pcr amplification, and with fragment cloning in the target-seeking carrier.5 ' end of every kind of Oligonucleolide primers of the synthetic arm that is used for increasing makes its different recognition sites that comprise rare cutting restriction enzyme, and it should be compatible with the cloning site of carrier polylinker and be present in the arm itself.With regard to GPR86, can design the primer of listing in the primer table hereinafter, 5 ' arm cloning site is Sal I/Not I and 3 ' arm cloning site is Asc I/Mfe I (Fig. 2 has shown the structure of used target-seeking carrier, comprises the relevant limit site).

Except that the arm primer to (5 ' armF/5 ' armR) and (3 ' armF/3 ' armR), for following purpose has designed the more primers special to the GPR86 locus: 5 ' and 3 ' probe primer to (5 ' prF/5 ' prR and 3 ' prF/3 ' are prR), be used to increase beyond each arm and extend to the 150-300bp fragment of two weak points of the non-duplicate factor group DNA beyond each arm, allow in the supposition target clone who separates, the target gene seat to be carried out Southern and analyze; Mouse genotyping primer is to (hetF and hetR), with the carrier special primer, be Asc350 when being used for multiplex PCR in this case, allow to distinguish wild type, heterozygosis and the mouse of isozygotying; With last target screening primer (3 ' scr), the downstream annealing of it and 3 ' arm district end, and with to carrier (TK5IBLMNL) 3 ' special primer of end, be Asc236 when pairing to produce the special 1.25kb amplimer (amplimer) of target incident in this case.The template DNA of the cell that this amplimer can only be changed by the genome wanted is derived, and its allows to identify the cell of correct target from clone's background of the carrier copy that comprises random integration.The position of these primers is shown in SEQ ID NO:21 with the genome structure that is used for the GPR86 locus zone of this target-seeking strategy.

Table 1:GPR86 primer sequence

musGPR86 5′pr F TATACATATGTTCAGCAGTACCAACTC-SEQ ID NO.8

musGPR86 5′pr R ACACCAGTGTATAGATAGCAAGAAGTC-SEQ ID NO.9

musGPR86 5′arm F cccgtcgacATGCTTTCTTTTATGACAAAATCCTTG-SEQ ID NO.10

musGPR86 5′arm R aaagcggccGcGAACAGCAGCTGTGTCATCCGAGTG-SEQ ID NO.11

musGPR86 3′arm F aaaggcgcgccAGGCAAGAACAGCAGGATCAAGCGAAG-SEQ ID NO.12

musGPR86 3′arm R aaacaattGTGGCTTCTGAGGCTATGGAAAGAGAG-SEQ ID NO.13

musGPR86 3′pr F ATATGGCACATTTGGTCCGCACTGCAC-SEQ ID NO.14

musGPR86 3′pr R GATGAGGAATGATGTCACACAGATGAG-SEQ ID NO.15

musGPR86 3′scr AAGGTCAAGATTAGCAAGTGATTCCAG-SEQ ID NO.16

musGPR86 hetF ATACCATACACTTACAGTCAAACCACC-SEQ ID NO.17

musGPR86 hetR GGTCTTCGCTTGATCCTGCTGTTCTTG-SEQ ID NO.18

Asc236 TTGGCTACCCGTGATATTGCTGAAGAG-SEQ ID NO.19

Asc350 GTCGTGACCCATGGCGATGCCTGCTTG-SEQ ID NO.20

Selection destroys the homology arm position of GPR86 gene from function.Preparation wherein waits to delete the target-seeking carrier that the GPR86 district replaces with non-homogeneous sequence, described non-homogeneous sequence is expressed reporter (reading independently lacZ gene of frame) and is selected box to constitute by endogenous gene, described reporter is positioned at selects the box upstream, described selection box is made of neomycin phosphotransferase (neo) gene that promotes, and its oriented is identical with the GPR86 gene.

In case with 5 ' and 3 ' homology arm be cloned among the target-seeking carrier TK5IBLMNL (referring to Fig. 2), utilize standard molecular biological technique to produce a large amount of highly purified DNA preparations.With the preparation of the restricted cutting fresh of the rare cutting restriction enzyme of another kind SwaI do not contain endotoxic 20 μ g DNA, cleavage site is the unique site between ampicillin resistance gene and the bacterium origin of replication in carrier framework.Precipitate linearizing DNA then, and be resuspended in 100 μ l phosphate buffered saline (PBS)s, prepare to carry out electroporation.

Behind the electroporation 24 hours, transfectional cell was cultivated 9 days in the nutrient culture media that contains 200 μ g/ml neomycins.The clone is chosen in 96 orifice plates, duplicating and increasing the back clone of between wherein endogenous GPR86 gene and the target-seeking construction homologous recombination having taken place that screens by PCR (utilizing primer 3 ' scr and Asc236 as mentioned above) to identify.The identification rate of positive colony can be 1-5%.These clones increase, thereby allow freezing duplicate, and the sufficient high quality DNA of preparation with the outside 5 that utilizes preparation as mentioned above ' and 3 ' probe confirm the target incident by the Southern trace, all use normal process (Russ et al, Nature2000 Mar 2; 404 (6773): 95-99).When with the Southern trace of the DNA of diagnostic restriction enzyme digestion during with external probe hybridization, the ES cell clone of the existence confirmation homology target by mutant band and unaltered wild type band.For example, when using 5 ' probe, the genomic DNA of HindIII digestion will produce the wild type band of 14.9kb and the target band of 12.0kb; And when using 3 ' probe, the DNA of Hind III cutting will produce the wild type band of 14.9kb and the target band of 7.0kb.

Embodiment 2: transgenosis GPR86 knocks out small white mouse: the generation of GPR86 deficient mice

Allow the mating of C57BL/6 male and female mouse, and separate blastocyst in the time of 3.5 days in gestation.Each blastocyst is injected 10-12 cell from selected clone, and 7-8 blastocyst is implanted the uterus of false pregnancy F1 jenny.The a brood of chimeric young mouse that has been born comprises the male mouse of several high levels (reaching 100%) depth striped (the fur color table of depth striped comes from the contribution of target clone's cell from tomorrow).Allow these male chimeras and female MF1 and 129 mouse mating, determine that by depth striped fur color with by pcr gene type somatotype kind of a system transmits respectively.

Use primer hetF and hetR and the third carrier special primer (Asc350) that the tail folder thing of dissolving is carried out pcr gene type somatotype.This multiplex PCR allows to produce the band of 207bp with primer hetF and hetR amplification wild type gene seat (if present).Deleted the site of hetF in the knock-out mice, therefore allelic amplification will be failed from target.But the Asc350 primer will combine with the hetR primer from the band of target gene seat amplification 380bp, wherein the hetR primer with just in time at the regional annealing of 3 ' arm inside.Therefore, the genotype of the young mouse of the following announcement of this multiplex PCR: the wild type sample shows the band of single 207bp; The hybrid DNA sample produces two bands of 207bp and 380bp; And homozygous sample will only show the target specific band of 380bp.

Embodiment 3: biological data: gene expression pattern RT-PCR

Utilize RT-PCR to show gene expression (Fig. 3) in liver and the leucocyte.

Embodiment 4: biological data: the GPR86 that RT-PCR measures expresses

Be used to cDNA storehouse, check the expression of GPR86 mRNA by RT-PCR from people and mouse.For the human sequence, available primer

Forward 5 '-GGTGTTTGTTCACATCCCCAGC-3 '

Reverse 5 '-TGGTGTTGCTTCCTTGTTGCTC-3 '

Produce the product of 364bp.

Reaction conditions is as follows:

85 ℃ of warm starts

94 ℃ 15 seconds

60 ℃ 30 seconds, 40 circulations

72 ℃ 60 seconds

4 ℃ of maintenances

Contain separated product on the Tris acetate EDTA Ago-Gel of ethidium bromide, and utilizing the UV light source to observe.

In marrow, thymus gland, lymph node, Gegenbaur's cell and cartilage cell, found the expression in people's derived tissues (Fig. 7) and cell.At two people's derived cells derived from the T cell is also to have found among Jurkat CD4+ and the Myla CD8+.Seeing low-level expression derived from lymphocytic Colo720 with in derived from monocytic THP1 cell.

In small white mouse tissue (Fig. 8), in spleen, salivary gland, spinal cord, tongue, fat, testis, heart, eye, lung, kidney, thymus gland, stomach and small intestine, brain, liver and gall-bladder, blood, bladder and adrenal gland, found expression.

The expression of GPR86 comprises derived from immune response cell, and GPR86 is relevant with immune cell function for this hint, and described immune cell function comprises the inflammatory that those relate to pain and the function of nerve aspect.

Embodiment 5:GPR86 knock-out mice stimulates to external world and the sensitivity tests of pain (analgesia test): the test of wagging the tail

The utilization analgesia meter analgesia test of implementing to wag the tail of wagging the tail.This equipment can use easily this method accurately and repeatably in rodent, measure pain sensitivity (D ' Amour, F.E.andD.L.Smith, 1941, Expt.Clin.Pharmacol., 16:179-184).This instrument is controlled lamp as thermal source with shadow shield.Lamp is positioned at below the animal so that the environment of not too restraining to be provided.Detect by automatic testing circuit and to wag the tail, but make user's hand free operant animal.Animal is limited in the ventilation duct, and its tail is placed on the sensing slot on equipment top.

The activation meeting that the opening that passes shadow shield arrives the strong beam of afterbody produces uncomfortable at some points, so the tail that animal will swing it leaves light beam.In automatic mode, photodetector detects the afterbody motion, causes that clock stops to close with shadow shield.The record shadow shield open and reaction of animals between T.T. of passing.

The wild-type mice of replying with age and gender matched of mutant transgenic mice can be compared.Single animal can be stood different heat settings is not more than 55 ℃ with generation afterbody temperature increase.

Test knock-out animal and wild type control-animal on the instrument of wagging the tail.Knock-out animal (6.88 ± 0.26sec) with the wild type of comparing contrast (5.95 ± 0.28) regain afterbody reaction time (latency) (between stimulate and and reaction between time) between there were significant differences.Therefore, knock-out animal is than wild type animal more insensitive (Fig. 4)

Embodiment 6:GPR86 knock-out mice stimulates to external world and the sensitivity tests of pain (analgesia test): the formalin test

The formalin thermometrically is to being expelled to the reaction of the noxious material in the rear solid end.5% formalin solution of 20 μ l volumes is subcutaneously injected into the back side of a rear solid end by thin gage needle.The actuating quantity that to lick, shake and sting rear solid end turns to accumulative total number second of being engaged in these behaviors.The service rating scale: the pawl of 1=injection of formalin rests on the base plate gently, bears less body weight; 2=lifts the pawl of injection; 3=licks, stings or shake the pawl of injection.

In the formalin test, see the reaction in two stages.Stage 1 begins after injection immediately, continues about 10 minutes, and representative is from the acute outburst of the activity of pain fiber.Stage 2 starts from about 20 minutes of injection back, continues about one hour.As if this stage is represented the reaction to tissue damage, comprises inflammatory hyperalgia.

Test GPR86 mutant in the formalin test according to the measurement of licking, stinging or shaking the number of injection foot, finds that replying of GPR86 mutant is less.Compare with the wild type oedema, the sufficient inflammatory edema of response formalin injection has also reduced by 12% (Fig. 5) in the KO mouse.

Embodiment 7:GPR86 knock-out mice stimulates to external world and the sensitivity tests of pain (analgesia test): the test of Von Frey filament

Von Frey filament has been used in the test of touching that is used for measuring threshold of pain.These filaments are calibration tinsels of one group of superfine specification.Measurement is to the threshold of recalling of mechanical stimulus.Animal stands on the scaoffold that the surface is wide gauge steel silk screen.From following insertion Von Frey filament, upwards by mesh, below the thorn rear solid end.When threshold value, mouse is reacted away from filament by the pawl of swinging it, next mentions pawl usually in addition, licks pawl and/or sounding.The machinery withdrawal threshold is defined as in three long run tests twice and causes that the minimum gauge tinsel of withdrawn reaction stimulates.

Test knock-out animal and wild type control-animal on electronics Vonfrey (each rear solid end is carried out 5 tests).(5.9 ± 0.2sec) recall with the wild type of comparing contrast (5.2 ± 0.2, p=0.03, t check) that there were significant differences between reaction time of pawl to knock-out animal.Therefore, knock-out animal is than wild type more insensitive (Fig. 6).

Embodiment 8: test GPR86 knocks out that small white mouse stimulates to external world and the susceptibility of pain (analgesia test): neuropathic pain

Can induce neuropathic pain (Kim and Chung1992) by the L5 spinal nerve of ligation anesthetized mice tightly.After the recovery, can measure the development of neuropathic pain and keep with regard to allodynia (to the pain perception of non-destructive stimulus) or hyperalgia (reaction to destructive stimulus increases).

Utilize von Frey filament (as described in example 7 above) to measure allodynia in during 4 weeks.Test each rear solid end, and relatively knock-out mice and the homonymy (damage side) of wild-type mice and the pawl reaction of offside (example is untreated).Can test hyperalgia with harmful heat, harmful harmful mechanical stimulus of cold-peace.Find all that in these two tests knock-out mice is more insensitive than the wild type contrast.

Apply for and patent for every that mentions in the presents, and quote in above-mentioned each part application and the patent or every part of file of reference, comprise between every application and patent suit time and quoting or every part of file (" file that application is quoted ") of reference, and every part the application and patent, and any manufacturer specification or the products catalogue of any product of quoting or mention in the file quoted of any application, all be collected herein by reference.And, the All Files of in this text, quoting, and quote in the file quoted of this text or the All Files of reference, and any manufacturer specification or the products catalogue of any product of quoting or mention in this text, all be collected herein by reference.

The various modifications of the method for the invention and system and variation are conspicuous to those skilled in the art, and do not run counter to scope and spirit of the present invention.Though described the present invention in conjunction with concrete preferred embodiment; it should be understood that claimed invention should unsuitablely not be limited to these specific embodiments, and can make many modifications and interpolation to these embodiments within the scope of the invention.In fact, the various modifications that are used to implement described mode of the present invention are significantly to the those of skill in the art of molecular biology or association area, and intention belongs in the scope of claim.And, can not depart from the scope of the present invention the various combinations of making the feature of dependent claims with the feature of independent claims.

Sequence table

SEQ ID NO：1

SEQ ID NO:1 has shown the cDNA sequence of people GPR86.

TCATTTGTAGGCTGAACTAATGACTGCCGCCATAAGAAGACAGAGAGAACTGAGTATCCTCCC

AAAGGTGACACTGGAAGCAATGAACACCACAGTGATGCAAGGCTTCAACAGATCTGAGCGGTG

CCCCAGAGACACTCGGATAGTACAGCTGGTATTCCCAGCCCTCTACACAGTGGTTTTCTTGACC

GGCATCCTGCTGAATACTTTGGCTCTGTGGGTGTTTGTTCACATCCCCAGCTCCTCCACCTTCAT

CATCTACCTCAAAAACACTTTGGTGGCCGACTTGATAATGACACTCATGCTTCCTTTCAAAATC

CTCTCTGACTCACACCTGGCACCCTGGCAGCTCAGAGCTTTTGTGTGTCGTTTTTCTTCGGTGAT

ATTTTATGAGACCATGTATGTGGGCATCGTGCTGTTAGGGCTCATAGCCTTTGACAGATTCCTC

AAGATCATCAGACCTTTGAGAAATATTTTTCTAAAAAAACCTGTTTTTGCAAAAACGGTCTCAA

TCTTCATCTGGTTCTTTTTGTTCTTCATCTCCCTGCCAAATATGATCTTGAGCAACAAGGAAGCA

ACACCATCGTCTGTGAAAAAGTGTGCTTCCTTAAAGGGGCCTCTGGGGCTGAAATGGCATCAA

ATGGTAAATAACATATGCCAGTTTATTTTCTGGACTGTTTTTATCCTAATGCTTGTGTTTTATGT

GGTTATTGCAAAAAAAGTATATGATTCTTATAGAAAGTCCAAAAGTAAGGACAGAAAAAACAA

CAAAAAGCTGGAAGGCAAAGTATTTGTTGTCGTGGCTGTCTTCTTTGTGTGTTTTGCTCCATTTC

ATTTTGCCAGAGTTCCATATACTCACAGTCAAACCAACAATAAGACTGACTGTAGACTGCAAAA

TCAACTGTTTATTGCTAAAGAAACAACTCTCTTTTTGGCAGCAACTAACATTTGTATGGATCCCT

TAATATACATATTCTTATGTAAAAAATTCACAGAAAAGCTACCATGTATGCAAGGGAGAAAGA

CCACAGCATCAAGCCAAGAAAATCATAGCAGTCAGACAGACAACATAACCTTAGGCTGACAAC

TGTACATAGGGTTAACTTCTATTTATTGATGAGACTTCCGTAGATAATGTGGAAATCAAATTTA

ACCAAGAAAAAAAGATTGGAACAAATGCTCTCTTACATTTTATTATCCTGGTGTACAGAAAAG

ATTATATAAAATTTAAATCCACATAGATCTATTCATAAGCTGAATGAACCATTACTAAGAGAAT

GCAACAGGATACAAATGGCCACTAGAGGTCATTATTTCTTTCTTTCTTTTTTTTTTTTTTAATTTC

AAGAGCATTTCACTTTAACATTTTGGAAAAGACTAAGGAGAAACGTATATCCCTACAAACCTCC

CCTCCAAACACCTTCTCACATTCTTTTCCACAATTCACATAACACTACTGCTTTTGTGCCCCTTA

AATGTAGATATGTGCTGAAAGAAAAAAAAAACGCCCAACTCTTGAAGTCCATTGCTGAAAACT

GCAGCCAGGGGTTGAAAGGGATGCAGACTTGAAGAGTCTGAGGAACTGAAGTGGGTCAGCAA

GACCTCTGAAATCCTGGGTAAAGGATTTTCTCCTTACAATTACAAACAGCCTCTTTCACATTAC

AATAATATACCATAGGAGGCACAAGCACCATTATTAAGCCACTTTGCTTACACCTTAAGTGTGT

ACAATTCAAGTGTGAGAATGCTGTGTTAACTATTCTTTGGAATTCTCCTTCTGTCCAGCAAATAC

TCTAATGATGGTTAAACATGGCACCTACTCAGCAATGCCTTCCTGGACCACAACCCCTATCCCC

CTGCCCCACCCTCCTCATTAAAAACAAATACTTCTACTGTTTGGGTGTGTGATAGGGTTCTCAAT

GCAGATCTCCCTTTTCTAGTTAGCTATATTCTTGACTGCATCCGCTAAAAATGTTAAAGCTTCTT

GAGAGACAGACATGCCAGATTTTCTTGGTATCTCCCATAATACGACCTACAGTCCATGGTCTAC

AGATGTTTTAAATAGAATTGCTATTCTCGATACATACAAAGACGTAATTGCTGACCCACAATCA

GTAACATCCATATTGGGAGATTTTTCAAAGGATGGTGACCCTGCTTGTATTTATTTACCTTGGTA

TTTTTTCTTGCATCCTTCTGTGATTCAAAAAAGTAAAATGTGGCTTTCTGAAATGATGGATAAGA

GTCTACATCTTCTAGAAAAAATACATAAAGGAGTAGTTAAGCTCTGTAAATGTGCCACGAGCTC

CAACACGACCATCGTAGGGTGAAGCCCACGTTTTCTTCCATGGCCTCAAAGGCCCTAGAACTTG

CCTACCTTTCTGGCCTTACCTCCTAGCTACTTATCCATCTCTTGAACTTTATACTCTTGTATAAAT

TTCTAACTTTCAGAAAATGCCATACTCTGTTTTGGCACCACACATGTATATTTCCCCCTGGTACA

CTTGGAAGACTCTTATCCATCTGTGAAACCCTATGTTGTCATCACTTGGTCCATGAAATATTACC

TGGCCAATATCCCACCATCACCTCAAACCCAATCACCCCCTCCTCTGTATGCTGTCACACCTATA

TTATTAAACTTATCACATTGCATTGTAATTACTTCCTGACCTTTGTATCTACTCTTTTAGTAACTG

ATGTATATATCTGAAAGGAGAGATTGTTTCATTGTGCAATCAATAAATGTTTGATAAAATAAAG

CCC

SEQ ID NO：2

SEQ ID NO:2 has shown the open read frame derived from SEQ ID NO:1.

ATGACTGCCGCCATAAGAAGACAGAGAGAACTGAGTATCCTCCCAAAGGTGACACTGGAAGCAATGAACACCACAGTGATG

CAAGGCTTCAACAGATCTGAGCGGTGCCCCAGAGACACTCGGATAGTACAGCTGGTATTCCCA

GCCCTCTACACAGTGGTTTTCTTGACCGGCATCCTGCTGAATACTTTGGCTCTGTGGGTGTTTGT

TCACATCCCCAGCTCCTCCACCTTCATCATCTACCTCAAAAACACTTTGGTGGCCGACTTGATAA

TGACACTCATGCTTCCTTTCAAAATCCTCTCTGACTCACACCTGGCACCCTGGCAGCTCAGAGCT

TTTGTGTGTCGTTTTTCTTCGGTGATATTTTATGAGACCATGTATGTGGGCATCGTGCTGTTAGG

GCTCATAGCCTTTGACAGATTCCTCAAGATCATCAGACCTTTGAGAAATATTTTTCTAAAAAAA

CCTGTTTTTGCAAAAACGGTCTCAATCTTCATCTGGTTCTTTTTGTTCTTCATCTCCCTGCCAAAT

ATGATCTTGAGCAACAAGGAAGCAACACCATCGTCTGTGAAAAAGTGTGCTTCCTTAAAGGGG

CCTCTGGGGCTGAAATGGCATCAAATGGTAAATAACATATGCCAGTTTATTTTCTGGACTGTTT

TTATCCTAATGCTTGTGTTTTATGTGGTTATTGCAAAAAAAGTATATGATTCTTATAGAAAGTCC

AAAAGTAAGGACAGAAAAAACAACAAAAAGCTGGAAGGCAAAGTATTTGTTGTCGTGGCTGTC

TTCTTTGTGTGTTTTGCTCCATTTCATTTTGCCAGAGTTCCATATACTCACAGTCAAACCAACAA

TAAGACTGACTGTAGACTGCAAAATCAACTGTTTATTGCTAAAGAAACAACTCTCTTTTTGGCA

GCAACTAACATTTGTATGGATCCCTTAATATACATATTCTTATGTAAAAAATTCACAGAAAAGC

TACCATGTATGCAAGGGAGAAAGACCACAGCATCAAGCCAAGAAAATCATAGCAGTCAGACA

GACAACATAACCTTAGGCTGA

SEQ ID NO：3

SEQ ID NO:3 has shown the amino acid sequence of people GPR86.

MTAAIRRQRELSILPKVTLEAMNTTVMQGFNRSERCPRDTRIVQLVFPALYTVVFLTGILLNTLALW

VFVHIPSSSTFIIYLKNTLVADLIMTLMLPFKILSDSHLAPWQLRAFVCRFSSVIFYETMYVGIVLLGLI

AFDRFLKIIRPLRNIFLKKPVFAKTVSIFIWFFLFFISLPNMILSNKEATPSSVKKCASLKGPLGLKWHQ

MVNNICQFIFWTVFILMLVFYVVIAKKVYDSYRKSKSKDRKNNKKLEGKVFVVVAVFFVCFAPFHF

ARVPYTHSQTNNKTDCRLQNQLFIAKETTLFLAATNICMDPLIYIFLCKKFTEKLPCMQGRKTTASS

QENHSSQTDNITLG

SEQ ID NO：4

SEQ ID NO:4 has shown the open read frame of mouse GPR86.

ATGCTCGGGACAATCAACACCACTGGGATGCAGGGCTTCAACAAGTCTGAGCGGTGCCCCAGG

GACACTCGGATGACACAGCTGCTGTTCCCGGTTCTCTATACTGTGGTCTTCCTGGCAGGCATCCT

GCTGAACACCGTGGCCCTCTGGGTGTTCGTCCACATCCCCAGCAATTCCACCTTTATCGTCTACC

TCAAGAACACTCTGGTGGCAGACTTGATAATGGCACTCATGCTGCCTTTCAAAATCCTTTCCGA

CTCACACCTTGCGCCCTGGCAGCTCCGAGGATTTGTGTGCACGCTCTCCTCCGTGGTCTTCTATG

AGACGATGTATGTGGGTATCATGATGCTGGGCCTCATCGCTTTCGACAGGTTCCTCAAGATCAT

CATGCCGTTCAGGAAAACCTTTGTCAAAAAGACGGCTTTCGCAAAAACAGTCTCCATTTCCGTC

TGGTCCCTGATGTTCTTCATCTCCCTGCCAAACATGATCTTGAACAAGGAGGCAACGCCATCAT

CCGTGAAGAAGTGTGCATCTTTGAAGAGTCCCCTTGGGCTGTGGTGGCATCAGGTGGTCAGTCA

CACCTGCCAGTTCATTTTCTGGGCTGTGTTTATTCTGATGCTTCTGTTTTATGCGGTGATTACCA

AAAAGGTGTACAACTCCTATAGGAAGTTTAGGAGTAAGGACAGCAGGCACAAGCGGCTGGAG

GTGAAGGTATTTATCGTCATGGCTGTCTTCTTTGTCTGCTTTGCCCCACTGCATTTTGTCAGAAT

ACCATACACTTACAGTCAAACCACCAATAAGACTGACTGTAGGTTAGAAAACCAGCTGTTTATT

GCTAAAGAAGCAACTCTCTTTCTGGCAACAACTAACATTTGTATGGACCCCTTAATATACATAA

TTTTATGCAAGAAGTTCACACAAAAGGTGCCATGTGTGAGATGGGGAAAGGCAAGAACAGCAG

GATCAAGCGAAGACCACCACAGTAGTCAGACAGACAACATCACCCTAGCCTGA

SEQ ID NO：5

SEQ ID NO:5 has shown the amino acid sequence of mouse GPR86.

MLGTINTTGMQGFNKSERCPRDTRMTQLLFPVLYTVVFLAGILLNTVALWVFVHIPSNSTFIVYLKN

TLVADLIMALMLPFKILSDSHLAPWQLRGFVCTLSSVVFYETMYVGIMMLGLIAFDRFLKIIMPFRK

TFVKKTAFAKTVSISVWSLMFFISLPNMILNKEATPSSVKKCASLKSPLGLWWHQVVSHTCQFIFWA

VFILMLLFYAVITKKVYNSYRKFRSKDSRHKRLEVKVFIVMAVFFVCFAPLHFVRIPYTYSQTTNKT

DCRLENQLFIAKEATLFLATTNICMDPLIYIILCKKFTQKVPCVRWGKARTAGSSEDHHSSQTDNITL

AZ

SEQ ID NO：6

SEQ ID NO:6 has shown the variable cDNA sequence of people GPR86.

TATGTTTATTGGTAACAGgtgacactggaagcaATGAACACCACAGTGATGCAAGGCTTCAACAGATCT

GAGCGGTGCCCCAGAGACACTCGGATAGTACAGCTGGTATTCCCAGCCCTCTACACAGTGGTTT

TCTTGACCGGCATCCTGCTGAATACTTTGGCTCTGTGGGTGTTTGTTCACATCCCCAGCTCCTCC

ACCTTCATCATCTACCTCAAAAACACTTTGGTGGCCGACTTGATAATGACACTCATGCTTCCTTT

CAAAATCCTCTCTGACTCACACCTGGCACCCTGGCAGCTCAGAGCTTTTGTGTGTCGTTTTTCTT

CGGTGATATTTTATGAGACCATGTATGTGGGCATCGTGCTGTTAGGGCTCATAGCCTTTGACAG

ATTCCTCAAGATCATCAGACCTTTGAGAAATATTTTTCTAAAAAAACCTGTTTTTGCAAAAACG

GTCTCAATCTTCATCTGGTTCTTTTTGTTCTTCATCTCCCTGCCAAATATGATCTTGAGCAACAA

GGAAGCAACACCATCGTCTGTGAAAAAGTGTGCTTCCTTAAAGGGGCCTCTGGGGCTGAAATG

GCATCAAATGGTAAATAACATATGCCAGTTTATTTTCTGGACTGTTTTTATCCTAATGCTTGTGT

TTTATGTGGTTATTGCAAAAAAAGTATATGATTCTTATAGAAAGTCCAAAAGTAAGGACAGAA

AAAACAACAAAAAGCTGGAAGGCAAAGTATTTGTTGTCGTGGCTGTCTTCTTTGTGTGTTTTGC

TCCATTTCATTTTGCCAGAGTTCCATATACTCACAGTCAAACCAACAATAAGACTGACTGTAGA

CTGCAAAATCAACTGTTTATTGCTAAAGAAACAACTCTCTTTTTGGCAGCAACTAACATTTGTA

TGGATCCCTTAATATACATATTCTTATGTAAAAAATTCACAGAAAAGCTACCATGTATGCAAGG

GAGAAAGACCACAGCATCAAGCCAAGAAAATCATAGCAGTCAGACAGACAACATAACCTTAG

GCTGACAACTGTACATAGGGTTAACTTCTATTTATTGATGAGACTTCCGTAGATAATGTGGAAA

TCAAATTTAACCAAGAAAAAAAGATTGGAACAAATGCTCTCTTACATTTTATTATCCTGGTGTA

CAGAAAAGATTATATAAAATTTAAATCCACATAGATCTATTCATAAGCTGAATGAACCATTACT

AAGAGAATGCAACAGGATACAAATGGCCACTAGAGGTCATTATTTCTTTCTTTCTTTTTTTTTTT

TTTAATTTCAAGAGCATTTCACTTTAACATTTTGGAAAAGACTAAGGAGAAACGTATATCCCTA

CAAACCTCCCCTCCAAACACCTTCTCACATTCTTTTCCACAATTCACATAACACTACTGCTTTTG

TGCCCCTTAAATGTAGATATGTGCTGAAAGAAAAAAAAAACGCCCAACTCTTGAAGTCCATTG

CTGAAAACTGCAGCCAGGGGTTGAAAGGGATGCAGACTTGAAGAGTCTGAGGAACTGAAGTG

GGTCAGCAAGACCTCTGAAATCCTGGGTAAAGGATTTTCTCCTTACAATTACAAACAGCCTCTT

TCACATTACAATAATATACCATAGGAGGCACAAGCACCATTATTAAGCCACTTTGCTTACACCT

TAAGTGTGTACAATTCAAGTGTGAGAATGCTGTGTTAACTATTCTTTGGAATTCTCCTTCTGTCC

AGCAAATACTCTAATGATGGTTAAACATGGCACCTACTCAGCAATGCCTTCCTGGACCACAACC

CCTATCCCCCTGCCCCACCCTCCTCATTAAAAACAAATACTTCTACTGTTTGGGTGTGTGATAGG

GTTCTCAATGCAGATCTCCCTTTTCTAGTTAGCTATATTCTTGACTGCATCCGCTAAAAATGTTA

AAGCTTCTTGAGAGACAGACATGCCAGATTTTCTTGGTATCTCCCATAATACGACCTACAGTCC

ATGGTCTACAGATGTTTTAAATAGAATTGCTATTCTCGATACATACAAAGACGTAATTGCTGAC

CCACAATCAGTAACATCCATATTGGGAGATTTTTCAAAGGATGGTGACCCTGCTTGTATTTATTT

ACCTTGGTATTTTTTCTTGCATCCTTCTGTGATTCAAAAAAGTAAAATGTGGCTTTCTGAAATGA

TGGATAAGAGTCTACATCTTCTAGAAAAAATACATAAAGGAGTAGTTAAGCTCTGTAAATGTG

CCACGAGCTCCAACACGACCATCGTAGGGTGAAGCCCACGTTTTCTTCCATGGCCTCAAAGGCC

CTAGAACTTGCCTACCTTTCTGGCCTTACCTCCTAGCTACTTATCCATCTCTTGAACTTTATACTC

TTGTATAAATTTCTAACTTTCAGAAAATGCCATACTCTGTTTTGGCACCACACATGTATATTTCC

CCCTGGTACACTTGGAAGACTCTTATCCATCTGTGAAACCCTATGTTGTCATCACTTGGTCCATG

AAATATTACCTGGCCAATATCCCACCATCACCTCAAACCCAATCACCCCCTCCTCTGTATGCTGT

CACACCTATATTATTAAACTTATCACATTGCATTGTAATTACTTCCTGACCTTTGTATCTACTCTT

TTAGTAACTGATGTATATATCTGAAAGGAGAGATTGTTTCATTGTGCAATCAATA AATGTTTGA

TAAAATAAAGCCC

SEQ ID NO：7

SEQ ID NO:7 has shown the variable amino acid sequence of people GPR86.

MNTTVMQGFNRSERCPRDTRIVQLVFPALYTVVFLTGILLNTLALWVFVHIPSSSTFIIYLKNTLVAD

LIMTLMLPFKILSDSHLAPWQLRAFVCRFSSVIFYETMYVGIVLLGLIAFDRFLKIIRPLRNIFLKKPVF

AKTVSIFIWFFLFFISLPNMILSNKEATPSSVKKCASLKGPLGLKWHQMVNNICQFIFWTVFILMLVF

YVVIAKKVYDSYRKSKSKDRKNNKKLEGKVFVVVAVFFVCFAPFHFARVPYTHSQTNNKTDCRLQ

NQLFIAKETTLFLAATNICMDPLIYIFLCKKFTEKLPCMQGRKTTASSQENHSSQTDNITLG

SEQ ID NO：8-20

SEQ ID NO:8-20 has shown and has knocked out the plasmid primer sequence.

TATACATATGTTCAGCAGTACCAACTC-SEQ ID NO.8

ACACCAGTGTATAGATAGCAAGAAGTC-SEQ ID NO.9

cccgtcgacATGCTTTCTTTTATGACAAAATCCTTG-SEQ ID NO.10

aaagcggccGcGAACAGCAGCTGTGTCATCCGAGTG-SEQ ID NO.11

aaaggcgcgccAGGCAAGAACAGCAGGATCAAGCGAAG-SEQ ID NO.12

aaacaattGTGGCTTCTGAGGCTATGGAAAGAGAG-SEQ ID NO.13

ATATGGCACATTTGGTCCGCACTGCAC-SEQ ID NO.14

GATGAGGAATGATGTCACACAGATGAG-SEQ ID NO.15

AAGGTCAAGATTAGCAAGTGATTCCAG-SEQ ID NO.16

ATACCATACACTTACAGTCAAACCACC-SEQ ID NO.17

GGTCTTCGCTTGATCCTGCTGTTCTTG-SEQ ID NO.18

TTGGCTACCCGTGATATTGCTGAAGAG-SEQ ID NO.19

GTCGTGACCCATGGCGATGCCTGCTTG-SEQ ID NO.20

SEQ ID NO：21

SEQ ID NO:21 has shown and has knocked out plasmid sequence.

>5′prF

|

| 2500

TTACAAATATGTTAAAACAAACATACAAGAGAACCTGAATACATATGTTCAGCAGTAACAACTCAAAAAGTCAAACAAATTAAATGTCCATCCGTAACAG

AATGTTTATACAATTTTGTTTGTATGTTCTCTTGGACTTATGTATACAAGTCGTCATTGTTGAGTTTTTCAGTTTGTTTAATTTACAGGTAGGCATTGTC

2600

CTTAACCAAAATATGGGAAAAGTTGTGTTTATCATTATGATCAGTGTCTATGTATTAACATAGACTAAGACAAAAATTAACACAGACTAAAATAAGCATG

GAATTGGTTTTATACCCTTTTCAACACAAATAGTAATACTAGTCACAGATACATAATTGTATCTGATTCTGTTTTTAATTGTGTCTGATTTTATTCGTAC

<5′prR

|

| 2700

AACACTTACACACTAGTATAAATATAAATGACTTCTTGCTATCTATACACTGGTGTTTAGAGTTTTCAAATAGTTGTTTTCTGTGTGTGTATGTTTTGTG

TTGTGAATGTGTGATCATATTTATATTTACTGAAGAACGATAGATATGTGACCACAAATCTCAAAAGTTTATCAACAAAACACACACACATACAAAACAC

2800

TGTGTGTGTGTGTGTGTGTGTGTATGTGTGTGTGTGTGCATGTCCCCTTGTGAGTGTGGGCCTGTGTACACTATCACACACATATAGGTGTCAAAGGACA

ACACACACACACACACACACACATACACACACACACACGTACAGGGGAACACTCACACCCGGACACATGTGATAGTGTGTGTATATCCACAGTTTCCTGT

2900

ACCTCAGGTGTCAGACATCATTTGTCATTTTGTTTGAGATAGAGTCTCCTTTTTACTGCTGCATATACCAGACCAACCACTCTGCATGCTTCTAAGGAAT

TGGAGTCCACAGTCTGTAGTAAACAGTAAAACAAACTCTATCTCAGAGGAAAAATGACGACGTATATGGTCTGGTTGGTGAGACGTACGAAGATTCCTTA

3000

TCTGTTTCCATATCCCCAATCTCGCAATACGAATGACAGAATTACATATACACATTACTGTGTCTACTTCTATATGGGCTCAGGAGATCTAAGTTTATTC

AGACAAAGGTATAGGGGTTAGAGCGTTATGCTTACTGTCTTAATGTATATGTGTAATGACACAGATGAAGATATACCCGAGTCCTCTAGATTCAAATAAG

>5′armF

|

>5′arm

|

| 3100

TTCCATGCTTGTGTGGCAAGTGCTTTACCTACTGCGTCATCTCCTATGTATTTCATCTTTAAAACATATGCTTTCTTTTATGACAAAATCCTTGTAACTA

AAGGTACGAACACACCGTTCACGAAATGGATGACGCAGTAGAGGATACATAAAGTAGAAATTTTGTATACGAAAGAAAATACTGTTTTAGGAACATTGAT

3200

AATATTAAGCATTTCAAGTATTGCTGTGAATATATTGCCTGTTTCTGAAGAGATTTTTCTAATACTGATTTTCACTTCAGGACATCTGCTTGTAAACTAC

TTATAATTCGTAAAGTTCATAACGACACTTATATAACGGACAAAGACTTCTCTAAAAAGATTATGACTAAAAGTGAAGTCCTGTAGACGAACATTTGATG

3300

ATAGTGTATTTAACTCATTATCACTTTTGGTTACCCTAATTGGAAAGTTTTAAAAAATTCACATGCTCTAAGGAAAGTACCTCAGTAGATTTCAGAGTAA

TATCACATAAATTGAGTAATAGTGAAAACCAATGGGATTAACCTTTCAAAATTTTTTAAGTGTACGAGATTCCTTTCATGGAGTCATCTAAAGTCTCATT

3400

ATAAGAGTTCTCTCCTAGGAATATTTGAGTTCCCACTTCAACTTACATCCTTAGTGAAAATGAAAGCAAACATCTCAACATTTCTAATGTTATTATATGA

TATTCTCAAGAGAGGATCCTTATAAACTCAAGGGTGAAGTTGAATGTAGGAATCACTTTTACTTTCGTTTGTAGAGTTGTAAAGATTACAATAATATACT

3500

TGCATGTTAACTACATCACCAGAAGTCCCTTTGCTCTTTGCCTTGATCCAGCTCAGGAATCCTGGAGGTCTAGCAAAGGAAGTAGGTGTAGGCAACTTCC

ACGTACAATTGATGTAGTGGTCTTCAGGGAAACGAGAAACGGAACTAGGTCGAGTCCTTAGGACCTCCAGATCGTTTCCTTCATCCACATCCGTTGAAGG

3600

ATTACAGACCAGTTTGTCCCATCTGACCATACTGGTTGGACAATTTACAAATTTAACCTTAGACCTGAGTGTGTACCAGACAGAACTGAGTGTCCGTTCA

TAATGTCTGGTCAAACAGGGTAGACTGGTATGACCAACCTGTTAAATGTTTAAATTGGAATCTGGACTCACACATGGTCTGTCTTGACTCACAGGCAAGT

3700

GCTTCTTTTTCCTGTATCAATCATTGACTCTTGGCATAAGGACTTCAGGATGAAGTGAACCACTCCAGCTGCTCTCTCAGGGGTGTGGTGGGGTTGGGAC

CGAAGAAAAAGGACATAGTTAGTAACTGAGAACCGTATTCCTGAAGTCCTACTTCACTTGGTGAGGTCGACGAGAGAGTCCCCACACCACCCCAACCCTG

3800

AAGGCAGGCTCTAAGTGCAAATTCTAGGGCCCAGTGGTAAGTTAGTGGTGGTCTCTATTACCACTATTTTGGGAAGGTGCTTAATTTCTTCATTTTGATT

TTCCGTCCGAGATTCACGTTTAAGATCCCGGGTCACCATTCAATCACCACCAGAGATAATGGTGATAAAACCCTTCCACGAATTAAAGAAGTAAAACTAA

3900

TTCACATCTAAAATAAGACTGGACTATGTTGTTATTGTAAGGGCTGTGATAACATAGTTACCTATAATACCATAGCTATTTTTATTTTATTACTTTGAGT

AAGTGTAGATTTTATTCTGACCTGATACAACAATAACATTCCCGACACTATTGTATCAATGGATATTATGGTATCGATAAAAATAAAATAATGAAACTCA

4000

CTATTCTTAGTGCATAAAAGAATTCCAGTCTTTAATTTATTCCAGTCTTTAAATTATTCAGCAGGCTCCTAAACACTGAAACTTTTCAATGCATGGGGTT

GATAAGAATCACGTATTTTCTTAAGGTCAGAAATTAAATAAGGTCAGAAATTTAATAAGTCGTCCGAGGATTTGTGACTTTGAAAAGTTACGTACCCCAA

4100

TTTTTGTTTGTTTGTTTGTTTGTTTGTTTTTGTTTGTTTTTTGTTTTTTAGTTCTTCCTTCCCGGCCTTCGTGAACTAATAAGCCCACAGTATTCCTATT

AAAAACAAACAAACAAACAAACAAACAAAAACAAACAAAAAACAAAAAATCAAGAAGGAAGGGCCGGAAGCACTTGATTATTCGGGTGTCATAAGGATAA

4200

TTCTTTTTCATTGACTGAGGAAGCTCGCATGACATCGTCCATGACAGGCCTCACTGTGAGCATATGATGGACGTTCTTTTACCCCTAACTATATGAAAAG

AAGAAAAAGTAACTGACTCCTTCGAGCGTACTGTAGCAGGTACTGTCCGGAGTGACACTCGTATACTACCTGCAAGAAAATGGGGATTGATATACTTTTC

4300

GACACTGTTGTGTATTTCATCATAAAAAGGGGGCAAGTATTTCAGAGTGAGTATAAATAACTTCCCAAATGAGGGGAACAAAAAGCGACAGGTGGACAAG

CTGTGACAACACATAAAGTAGTATTTTTCCCCCGTTCATAAAGTCTCACTCATATTTATTGAAGGGTTTACTCCCCTTGTTTTTCGCTGTCCACCTGTTC

4400

CCCTTGGAGCTACGGCCCGCAGCTTGGAGATGGCACTTCCACACAGGCCTAGGGAGCAGCGTGCAGAGAGGCCCTTCCAAGAGGAACAGGCTTTCCAACA

GGGAACCTCGATGCCGGGCGTCGAACCTCTACCGTGAAGGTGTGTCCGGATCCCTCGTCGCACGTCTCTCCGGGAAGGTTCTCCTTGTCCGAAAGGTTGT

4500

AATATCAGCTAAGGAACTTACCAAGGGGAGCTCTCATTTAACAAGTGTGTAGTAGTTATGAAGATGACTCAGCTAGAGAAGACCAACCATGACCATGCAG

TTATAGTCGATTCCTTGAATGGTTCCCCTCGAGAGTAAATTGTTCACACATCATCAATACTTCTACTGAGTCGATCTCTTCTGGTTGGTACTGGTACGTC

4600

AGCTCAATCCATGAAACCCACATGTTGGAAAGATATAACTAACCCACTGAAGGAAAACACACACACACATACACACACACTCACACACTCACAATAAATA

TCGAGTTAGGTACTTTGGGTGTACAACCTTTCTATATTGATTGGGTGACTTCCTTTTGTGTGTGTGTGTATGTGTGTGTGAGTGTGTGAGTGTTATTTAT

4700

TGTATTTCACAACCAAAAGAATGTACCTTTGTGACCCTAGAAATGTCCATTGCTGATCAGTTCCTCTTTTCAAAGTTTCCCACATTTGGCATTTTGAAAT

ACATAAAGTGTTGGTTTTCTTACATGGAAACACTGGGATCTTTACAGGTAACGACTAGTCAAGGAGAAAAGTTTCAAAGGGTGTAAACCGTAAAACTTTA

4800

TTGACCAAAATGTGATTTCTACTTCTCATATTATGAAATTATCCTGTACAGAATTTCCCTTGTTTAAAGTAAAAATTTCCAGACCTTCAATGTTATATTG

AACTGGTTTTACACTAAAGATGAAGAGTATAATACTTTAATAGGACATGTCTTAAAGGGAACAAATTTCATTTTTAAAGGTCTGGAAGTTACAATATAAC

4900

GATTGGGGGCTATTGAGAAATTTAAAATTGACTGTGTTTATGGTTAAAATTAACAATAAACCTACTTTAAAATATTGATTTTGCTAGGTTTTTATCCTCT

CTAACCCCCGATAACTCTTTAAATTTTAACTGACACAAATACCAATTTTAATTGTTATTTGGATGAAATTTTATAACTAAAACGATCCAAAAATAGGAGA

5000

TATAAAGCAAAAGGATATATATTTAAGTTAAGGGGCATTACATCAGTAAATCCTAACATATTTTAGCCAAAAAGAAAATTTTTACAATGTGCCATTTATT

ATATTTCGTTTTCCTATATATAAATTCAATTCCCCGTAATGTAGTCATTTAGGATTGTATAAAATCGGTTTTTCTTTTAAAAATGTTACACGGTAAATAA

5100

CCCTAGAGTTAACCAACGTTCCCTGGAAACACAGTACATTTCATTTTAAAATGTTCAAATGTGGAATCAATGGATTCCAAATTCTCAACCAAGTAATATA

GGGATCTCAATTGGTTGCAAGGGACCTTTGTGTCATGTAAAGTAAAATTTTACAAGTTTACACCTTAGTTACCTAAGGTTTAAGAGTTGGTTCATTATAT

5200

GGAATTACTAGTTAGCCAAACCCTGAAGACGGTCCATCTCTACGGTAAGCGCGTGGCAAGAACGGTGAAGTGAAACTTGCCATAGAGAACCACACATCCT

CCTTAATGATCAATCGGTTTGGGACTTCTGCCAGGTAGAGATGCCATTCGCGCACCGTTCTTGCCACTTCACTTTGAACGGTATCTCTTGGTGTGTAGGA

5300

CCCCAGGATCATCTGATTTCCTCTTTCCCTTCATCTGTCGGGCAGGGAAGCAGACTGTCACACATTGTAACTTTCTGCACACTCCCTGAGATTTAAAACG

GGGGTCCTAGTAGACTAAAGGAGAAAGGGAAGTAGACAGCCCGTCCCTTCGTCTGACAGTGTGTAACATTGAAAGACGTGTGAGGGACTCTAAATTTTGC

5400

AAACACTGTACCAATCTGAGGCCAGCCCTGATTCAAAACTTCTCTAAGTCTCAAGAATGGAGGTGGTTTCCCAAAAGGGCTTCCTAAAAGTGCCACACTG

TTTGTGACATGGTTAGACTCCGGTCGGGACTAAGTTTTGAAGAGATTCAGAGTTCTTACCTCCACCAAAGGGTTTTCCCGAAGGATTTTCACGGTGTGAC

5500

TAGCTCCACTCAAACTGGGCAATTCAAGGAACCCAAGCAATGAGCGGCTGGCATGTTTAGCTTTCACTGATGTGCCCTGGGTCGGTCCATCGGTCCCCCT

ATCGAGGTGAGTTTGACCCGTTAAGTTCCTTGGGTTCGTTACTCGCCGACCGTACAAATCGAAAGTGACTACACGGGACCCAGCCAGGTAGCCAGGGGGA

5600

CCCCCACACTCCTATGAAGTAGATGATGATTTCAAACTTGCTACATGAGAGAATGAGGTATATAGCTCTTAAAACATTTGTTTAAACATAATACCGAATA

GGGGGTGTGAGGATACTTCATCTACTACTAAAGTTTGAACGATGTACTCTCTTACTCCATATATCGAGAATTTTGTAAACAAATTTGTATTATGGCTTAT

5700

ATGCAGAGGTCCAGGAATAATAGCTACCACTTCTTGGGAAATGTGACTCATAATCTGCCACAGATCTAGGGATCAAAAGACTAACAGCAATGTCTGAAGA

TACGTCTCCAGGTCCTTATTATCGATGGTGAAGAACCCTTTACACTGAGTATTAGACGGTGTCTAGATCCCTAGTTTTCTGATTGTCGTTACAGACTTCT

5800

AAGGAAACAAAGCAAAACAAACAAGCAACCAACAACCAAAGCCTAGCCTTAGACATACAAACTTCTTGCACGCCCAGGCTGTAAGGGACAGAAGATGATT

TTCCTTTGTTTCGTTTTGTTTGTTCGTTGGTTGTTGGTTTCGGATCGGAATCTGTATGTTTGAAGAACGTGCGGGTCCGACATTCCCTGTCTTCTACTAA

5900

TGATTTTCAGATCAAGTCTTCTCCTCCTACTTGATTGTAATTTCATACTATTTTCTTCATCATTTTAACTCCTACTATTACATATGAAGTATTCAATAAG

ACTAAAAGTCTAGTTCAGAAGAGGAGGATGAACTAACATTAAAGTATGATAAAAGAAGTAGTAAAATTGAGGATGATAATGTATACTTCATAAGTTATTC

6000

GTCGCTGAATGTGAATAATAAAAAGTAAAACACTGGGCCTAGAGAAATAGTTCGGCGGTTAAAGGGATTTTCCTGGCACCCACTCCAGGCAGCTCACAGT

CAGCGACTTACACTTATTATTTTTCATTTTGTGACCCGGATCTCTTTATCAAGCCGCCAATTTCCCTAAAAGGACCGTGGGTGAGGTCCGTCGAGTGTCA

6100

CACCTTGGACCCCAGCTCCAGGGAATCCAACACTTCTGGCCTCTGAGGGCACCTGAATGCATATGCGCCCTTACACATATAATTTAATATAATAAAATAG

GTGGAACCTGGGGTCGAGGTCCCTTAGGTTGTGAAGACCGGAGACTCCCGTGGACTTACGTATACGCGGGAATGTGTATATTAAATTATATTATTTTATC

6200

ACCACATGTTAAGAATATGGCGTGGTGGCGTGCCTGCAGAGCCGGTGACAGCACTGGATTCAATACTCAGAACACGCAACCAATAACTACTGGTAAATGC

TGGTGTACAATTCTTATACCGCACCACCGCACGGACGTCTCGGCCACTGTCGTGACCTAAGTTATGAGTCTTGTGCGTTGGTTATTGATGACCATTTACG

6300

TCCAAAGGTCTAAAGACAAAGAGGTTCTAGAAAATGTGGGTATTACAGAAGCCCACAAGGGGAAATGAGGAAGGAAAAGCAGTATAAGAAAGCAGAACCT

AGGTTTCCAGATTTCTGTTTCTCCAAGATCTTTTACACCCATAATGTCTTCGGGTGTTCCCCTTTACTCCTTCCTTTTCGTCATATTCTTTCGTCTTGGA

6400

TTACTTCACAAGCTATTCGTGGGTTGAGCTAGTAACTGCCACAACTCATACAGCTGAGTCTCTTCCAAAACAAAGGTCAGTTTAATTTTTCTTATGAATT

AATGAAGTGTTCGATAAGCACCCAACTCGATCATTGACGGTGTTGAGTATGTCGACTCAGAGAAGGTTTTGTTTCCAGTCAAATTAAAAAGAATACTTAA

_____________________EXON 1____________________________>

6500

TGCTTGCTATAAAAGGTAAAAGGTGGTTAAATTTTCTAGAAATCTAAATATAATATTCTGTAATCACGGGCTGCAAGCATAAGGTACTTCATAACGCATG

ACGAACGATATTTTCCATTTTCCACCAATTTAAAAGATCTTTAGATTTATATTATAAGACATTAGTGCCCGACGTTCGTATTCCATGAAGTATTGCGTAC

6600

ACACACTACCCTGTACTCTTACATCTTAAATTAGGTTCTGTCACCGCCGTGTAGTGATTGGTGCTTGCTATTTGGACTTCACAAGGGTAGACATTAACAG

TGTGTGATGGGACATGAGAATGTAGAATTTAATCCAAGACAGTGGCGGCACATCACTAACCACGAACGATAAACCTGAAGTGTTCCCATCTGTAATTGTC

6700

GACTTTATAAAACAAAACTAAACTGAACAAAAACCAACCAAACAACAACAGCAACAAAACTAAGCTATGTGCTCAAAAAAGCTCTAGCCAAAGAATCAAA

CTGAAATATTTTGTTTTGATTTGACTTGTTTTTGGTTGGTTTGTTGTTGTCGTTGTTTTGATTCGATACACGAGTTTTTTCGAGATCGGTTTCTTAGTTT

6800

TCCGCTGAATGAGTTAAATAGTAAGTTTGTTTTTACAAGAAGTAGTTATCTGCCAATCCACAAGGACCCTTTGTTTACCTTCCTTTGTATGCCTGCTTAT

AGGCGACTTACTCAATTTATCATTCAAACAAAAATGTTCTTCATCAATAGACGGTTAGGTGTTCCTGGGAAACAAATGGAAGGAAACATACGGACGAATA

>ATG

|

| 6900

GTGTAATCTTTCTCTGTGTGCATGTTTATGTGTAATAGCTGATGCTCGGGACAATCAACACCACTGGGATGCAGGGCTTCAACAAGTCTGAGCGGTGCCC

CACATTAGAAAGAGACACACGTACAAATACACATTATCGACTACGAGCCCTGTTAGTTGTGGTGACCCTACGTCCCGAAGTTGTTCAGACTCGCCACGGG

M L G T I N T T G M Q G F N K S E R C P>

________________________MUSGPR86__________________________>

____________________________EXON 2_____________________________>

<5′armR >7tm

| |

| | 7000

CAGGGACACTCGGATGACACAGCTGCTGTTCCCGGTTCTCTATACTGTGGTCTTCCTGGCAGGCATCCTGCTGAACACCGTGGCCCTCTGGGTGTTCGTC

GTCCCTGTGAGCCTACTGTGTCGACGACAAGGGCCAAGAGATATGACACCAGAAGGACCGTCCGTAGGACGACTTGTGGCACCGGGAGACCCACAAGCAG

R D T R M T O L L F P V L Y T V V F L A G I L L N T V A L W V F V>

_____________________________________________MUSGPR86______________________________________________>

______________________________________________EXON 2________________________________________________>

7100

CACATCCCCAGCAATTCCACCTTTATCGTCTACCTCAAGAACACTCTGGTGGCAGACTTGATAATGGCACTCATGCTGCCTTTCAAAATCCTTTCCGACT

GTGTAGGGGTCGTTAAGGTGGAAATAGCAGATGGAGTTCTTGTGAGACCACCGTCTGAACTATTACCGTGAGTACGACGGAAAGTTTTAGGAAAGGCTGA

H I P S N S T F I V Y L K N T L V A D L I M A L M L P F K I L S D>

_____________________________________________MUSGPR86______________________________________________>

______________________________________________EXON 2________________________________________________>

7200

CACACCTTGCGCCCTGGCAGCTCCGAGGATTTGTGTGCACGCTCTCCTCCGTGGTCTTCTATGAGACGATGTATGTGGGTATCATGATGCTGGGCCTCAT

GTGTGGAACGCGGGACCGTCGAGGCTCCTAAACACACGTGCGAGAGGAGGCACCAGAAGATACTCTGCTACATACACCCATAGTACTACGACCCGGAGTA

S H L A P W Q L R G F V C T L S S V V F Y E T M Y V G I M M L G L I>

_____________________________________________MUSGPR86______________________________________________>

______________________________________________EXON 2________________________________________________>

7300

CGCTTTCGACAGGTTCCTCAAGATCATCATGCCGTTCAGGAAAACCTTTGTCAAAAAGACGGCTTTCGCAAAAACAGTCTCCATTTCCGTCTGGTCCCTG

GCGAAAGCTGTCCAAGGAGTTCTAGTAGTACGGCAAGTCCTTTTGGAAACAGTTTTTCTGCCGAAAGCGTTTTTGTCAGAGGTAAAGGCAGACCAGGGAC

A F D R F L K I I M P F R K T F V K K T A F A K T V S I S V W S L>

_____________________________________________MUSGPR86______________________________________________>

______________________________________________EXON 2________________________________________________>

7400

ATGTTCTTCATCTCCCTGCCAAACATGATCTTGAACAAGGAGGCAACGCCATCATCCGTGAAGAAGTGTGCATCTTTGAAGAGTCCCCTTGGGCTGTGGT

TACAAGAAGTAGAGGGACGGTTTGTACTAGAACTTGTTCCTCCGTTGCGGTAGTAGGCACTTCTTCACACGTAGAAACTTCTCAGGGGAACCCGACACCA

M F F I S L P N M I L N K E A T P S S V K K C A S L K S P L G L W>

_____________________________________________MUSGPR86______________________________________________>

______________________________________________EXON 2________________________________________________>

7500

GGCATCAGGTGGTCAGTCACACCTGCCAGTTCATTTTCTGGGCTGTGTTTATTCTGATGCTTCTGTTTTATGCGGTGATTACCAAAAAGGTGTACAACTC

CCGTAGTCCACCAGTCAGTGTGGACGGTCAAGTAAAAGACCCGACACAAATAAGACTACGAAGACAAAATACGCCACTAATGGTTTTTCCACATGTTGAG

W H Q V V S H T C Q F I F W A V F I L M L L F Y A V I T K K V Y N S>

_____________________________________________MUSGPR86______________________________________________>

______________________________________________EXON 2________________________________________________>

7600

CTATAGGAAGTTTAGGAGTAAGGACAGCAGGCACAAGCGGCTGGAGGTGAAGGTATTTATCGTCATGGCTGTCTTCTTTGTCTGCTTTGCCCCACTGCAT

GATATCCTTCAAATCCTCATTCCTGTCGTCCGTGTTCGCCGACCTCCACTTCCATAAATAGCAGTACCGACAGAAGAAACAGACGAAACGGGGTGACGTA

Y R K F R S K D S R H K R L E V K V F I V M A V F F V C F A P L H>

_____________________________________________MUSGPR86______________________________________________>

______________________________________________EXON 2________________________________________________>

>hetF

|

| 7700

TTTGTCAGAATACCATACACTTACAGTCAAACCACCAATAAGACTGACTGTAGGTTAGAAAACCAGCTGTTTATTGCTAAAGAAGCAACTCTCTTTCTGG

AAACAGTCTTATGGTATGTGAATGTCAGTTTGGTGGTTATTCTGACTGACATCCAATCTTTTGGTCGACAAATAACGATTTCTTCGTTGAGAGAAAGACC

F V R I P Y T Y S Q T T N K T D C R L E N Q L F I A K E A T L F L>

_____________________________________________MUSGPR86______________________________________________>

______________________________________________EXON 2________________________________________________>

>3′armF

|

>3′arm

|

| 7800

CAACAACTAACATTTGTATGGACCCCTTAATATACATAATTTTATGCAAGAAGTTCACACAAAAGGTGCCATGTGTGAGATGGGGAAAGGCAAGAACAGC

GTTGTTGATTGTAAACATACCTGGGGAATTATATGTATTAAAATACGTTCTTCAAGTGTGTTTTCCACGGTACACACTCTACCCCTTTCCGTTCTTGTCG

A T T N I C M D P L I Y I I L C K K F T Q K V P C V R W G K A R T A>

_____________________________________________MUSGPR86______________________________________________>

______________________________________________EXON 2________________________________________________>

<hetR >Stop

| |

| | 7900

AGGATCAAGCGAAGACCACCACAGTAGTCAGACAGACAACATCACCCTAGCCTGACCACTGTGTCCCACAGGTTAATTTCACGCATGGCCTCACGTCTAT

TCCTAGTTCGCTTCTGGTGGTGTCATCAGTCTGTCTGTTGTAGTGGGATCGGACTGGTGACACAGGGTGTCCAATTAAAGTGCGTACCGGAGTGCAGATA

G S S E D H H S S Q T D N I T L A *>

______________________MUSGPR86________________________>

______________________________________________EXON 2________________________________________________>

8000

TTATGGATGGGATTTCAAAAGATCATTTATGTGGAGACCTCATTTAAGCATTACAGGAAAAAAGAGGGGAACAAACAGTTCCCTACATTTTATTATCCTA

AATACCTACCCTAAAGTTTTCTAGTAAATACACCTCTGGAGTAAATTCGTAATGTCCTTTTTTCTCCCCTTGTTTGTCAAGGGATGTAAAATAATAGGAT

______________________________________________EXON 2________________________________________________>

8100

GTGTATGGAAAAACATTATGCCCATTTTAACCACATAGACGTATTTATAAGCAGGATAAATTAAGAGACCATGTAATACAGCAAATGGCCACTAGATGTC

CACATACCTTTTTGTAATACGGGTAAAATTGGTGTATCTGCATAAATATTCGTCCTATTTAATTCTCTGGTACATTATGTCGTTTACCGGTGATCTACAG

______________________________________________EXON 2________________________________________________>

8200

ACCTTTTCAAGGGCATTCATGTACTATGGAAAAGGTTAATGGGAAACAGGTTTGCCTGAAAAATCTTCCTTCTAGTTACCACCCCACCATCTCTTCACAC

TGGAAAAGTTCCCGTAAGTACATGATACCTTTTCCAATTACCCTTTGTCCAAACGGACTTTTTAGAAGGAAGATCAATGGTGGGGTGGTAGAGAAGTGTG

______________________________________________EXON 2________________________________________________>

8300

ATATATTCCCTAAAACACCAGGCTGGCTTTTACAGCCTTCAGAATGCTGACACTTGTGAACAGAAACCAACCAACTTGCATATCCAGTGCCTGTGTGGAA

TATATAAGGGATTTTGTGGTCCGACCGAAAATGTCGGAAGTCTTACGACTGTGAACACTTGTCTTTGGTTGGTTGAACGTATAGGTCACGGACACACCTT

______________________________________________EXON 2________________________________________________>

8400

AGGCTAAGGTGGGGGCTCAAAGAGATGCAGTCTGAGGAACCAAAGTGGGTTGGTCAAAATAACCCCAGGCATCTCAAAAGATTTCCTCCTTACAAGTGCA

TCCGATTCCACCCCCGAGTTTCTCTACGTCAGACTCCTTGGTTTCACCCAACCAGTTTTATTGGGGTCCGTAGAGTTTTCTAAAGGAGGAATGTTCACGT

______________________________________________EXON 2________________________________________________>

8500

AAGGGCTGCTCCTACATCTAAACAGAGCACCAGAAGAGAGGCACATGCAACAGGCAAAGCCAGTTCACAGCCATGTGCAATCCAGAGAGGGGAAGTGTTT

TTCCCGACGAGGATGTAGATTTGTCTCGTGGTCTTCTCTCCGTGTACGTTGTCCGTTTCGGTCAAGTGTCGGTACACGTTAGGTCTCTCCCCTTCACAAA

______________________________________________EXON 2________________________________________________>

8600

AGTCAGCAAAACCTTCCTGGGCAACAGCATCTGCATCCTGTTGGAAAATACTTATTTCCCTCACTGTTTATCTATAAATTAGGTTCCTTGCTACACACTG

TCAGTCGTTTTGGAAGGACCCGTTGTCGTAGACGTAGGACAACCTTTTATGAATAAAGGGAGTGACAAATAGATATTTAATCCAAGGAACGATGTGTGAC

______________________________________________EXON 2________________________________________________>

8700

TCTTATATTAACTGCTTTCATTCTTAGCCACATTTCCCAAAAACAGGGTTCGTAAAAAGACAGCAAAATCACACATTTTTACAAAAGAAATGGGGTAAGG

AGAATATAATTGACGAAAGTAAGAATCGGTGTAAAGGGTTTTTGTCCCAAGCATTTTTCTGTCGTTTTAGTGTGTAAAAATGTTTTCTTTACCCCATTCC

______________________________________________EXON 2________________________________________________>

8800

ATATCCTAGACGGGATGTTTGTTGTACACTATCCTTAGTGCATGTGAGCAAGGGGATGGTTGGCCTGGCATTAGTAATATTCATGTGGGAAGATTTTTCA

TATAGGATCTGCCCTACAAACAACATGTGATAGGAATCACGTACACTCGTTCCCCTACCAACCGGACCGTAATCATTATAAGTACACCCTTCTAAAAAGT

______________________________________________EXON 2________________________________________________>

8900

AAGCCTGATTCATTTTATTTGGGCCTATCTACTTCCCTGGATCTCATTATGGGTTTAAGAAAATTAAAATATGTGGCTGGTGATGCTGGGTTTTCTGGAG

TTCGGACTAAGTAAAATAAACCCGGATAGATGAAGGGACCTAGAGTAATACCCAAATTCTTTTAATTTTATACACCGACCACTACGACCCAAAAGACCTC

______________________________________________EXON 2________________________________________________>

<3′armR

|

| 9000

AACACGACGACTCTTCCCCTAACCGCGCCTGTAATGTGAAATCCTGGCTTCTCTCTTTCCATAGCCTCAGAAGCCACAGAGGCAAGAGAACTTCCTTGTC

TTGTGCTGCTGAGAAGGGGATTGGCGCGGACATTACACTTTAGGACCGAAGAGAGAAAGGTATCGGAGTCTTCGGTGTCTCCGTTCTCTTGAAGGAACAG

______________________________________________EXON 2________________________________________________>

<3′scr >3′prF

| |

| | 9100

TTCCTGGAATCACTTGCTAATCTTGACCTTTACAAACTCCATTTAATATGGCACATTTGGTCCGCACTGCACTGTACATAGAACCTTCCATCTGACCCAC

AAGGACCTTAGTGAACGATTAGAACTGGAAATGTTTGAGGTAAATTATACCGTGTAAACCAGGCGTGACGTGACATGTATCTTGGAAGGTAGACTGGGTG

______________________________________________EXON 2________________________________________________>

9200

TTTGTAAGTTCCTATTGATTAGTGACACCCAAAGTTGCTGTCCCTTACTCCAGGTAACCTTACCTGCCCAACCTCCTCCGAGTCCTCCAGCCCAGTCACT

AAACATTCAAGGATAACTAATCACTGTGGGTTTCAACGACAGGGAATGAGGTCCATTGGAATGGACGGGTTGGAGGAGGCTCAGGAGGTCGGGTCAGTGA

______________________________________________EXON 2________________________________________________>

<3′prR >polyA

| |

| | 9300

TCCTCATCTGTGTGACATCATTCCTCATCTACTTATCGAATTGATTTAGGATAATTTGCTAAATATGCACTGTGCAATTAATAAATTTTGCTAAAACAAA

AGGAGTAGACACACTGTAGTAAGGAGTAGATGAATAGCTTAACTAAATCCTATTAAACGATTTATACGTGACACGTTAATTATTTAAAACGATTTTGTTT

_______________________________________EXON 2________________________________________>

Sequence table

＜110〉Paradim Medical Treatment Co., Ltd (Paradigm Therapeutics Limited)

＜120〉purposes of the receptor GPR 86

<130>PO20634GBR

<150>GB0414798.9

<151>2004-07-01

<150>GB0510253.8

<151>2005-05-19

<150>US 60/683,471

<151>2005-05-20

<150>US 60/586,513

<151>2004-07-09

<160>21

<170>PatentIn version 3.3

<210>1

<211>2766

<212>DNA

＜213〉human (Homo sapiens)

<400>1

tcatttgtag gctgaactaa tgactgccgc cataagaaga cagagagaac tgagtatcct 60

cccaaaggtg acactggaag caatgaacac cacagtgatg caaggcttca acagatctga 120

gcggtgcccc agagacactc ggatagtaca gctggtattc ccagccctct acacagtggt 180

tttcttgacc ggcatcctgc tgaatacttt ggctctgtgg gtgtttgttc acatccccag 240

ctcctccacc ttcatcatct acctcaaaaa cactttggtg gccgacttga taatgacact 300

catgcttcct ttcaaaatcc tctctgactc acacctggca ccctggcagc tcagagcttt 360

tgtgtgtcgt ttttcttcgg tgatatttta tgagaccatg tatgtgggca tcgtgctgtt 420

agggctcata gcctttgaca gattcctcaa gatcatcaga cctttgagaa atatttttct 480

aaaaaaacct gtttttgcaa aaacggtctc aatcttcatc tggttctttt tgttcttcat 540

ctccctgcca aatatgatct tgagcaacaa ggaagcaaca ccatcgtctg tgaaaaagtg 600

tgcttcctta aaggggcctc tggggctgaa atggcatcaa atggtaaata acatatgcca 660

gtttattttc tggactgttt ttatcctaat gcttgtgttt tatgtggtta ttgcaaaaaa 720

agtatatgat tcttatagaa agtccaaaag taaggacaga aaaaacaaca aaaagctgga 780

aggcaaagta tttgttgtcg tggctgtctt ctttgtgtgt tttgctccat ttcattttgc 840

cagagttcca tatactcaca gtcaaaccaa caataagact gactgtagac tgcaaaatca 900

actgtttatt gctaaagaaa caactctctt tttggcagca actaacattt gtatggatcc 960

cttaatatac atattcttat gtaaaaaatt cacagaaaag ctaccatgta tgcaagggag 1020

aaagaccaca gcatcaagcc aagaaaatca tagcagtcag acagacaaca taaccttagg 1080

ctgacaactg tacatagggt taacttctat ttattgatga gacttccgta gataatgtgg 1140

aaatcaaatt taaccaagaa aaaaagattg gaacaaatgc tctcttacat tttattatcc 1200

tggtgtacag aaaagattat ataaaattta aatccacata gatctattca taagctgaat 1260

gaaccattac taagagaatg caacaggata caaatggcca ctagaggtca ttatttcttt 1320

ctttcttttt ttttttttta atttcaagag catttcactt taacattttg gaaaagacta 1380

aggagaaacg tatatcccta caaacctccc ctccaaacac cttctcacat tcttttccac 1440

aattcacata acactactgc ttttgtgccc cttaaatgta gatatgtgct gaaagaaaaa 1500

aaaaacgccc aactcttgaa gtccattgct gaaaactgca gccaggggtt gaaagggatg 1560

cagacttgaa gagtctgagg aactgaagtg ggtcagcaag acctctgaaa tcctgggtaa 1620

aggattttct ccttacaatt acaaacagcc tctttcacat tacaataata taccatagga 1680

ggcacaagca ccattattaa gccactttgc ttacacctta agtgtgtaca attcaagtgt 1740

gagaatgctg tgttaactat tctttggaat tctccttctg tccagcaaat actctaatga 1800

tggttaaaca tggcacctac tcagcaatgc cttcctggac cacaacccct atccccctgc 1860

cccaccctcc tcattaaaaa caaatacttc tactgtttgg gtgtgtgata gggttctcaa 1920

tgcagatctc ccttttctag ttagctatat tcttgactgc atccgctaaa aatgttaaag 1980

cttcttgaga gacagacatg ccagattttc ttggtatctc ccataatacg acctacagtc 2040

catggtctac agatgtttta aatagaattg ctattctcga tacatacaaa gacgtaattg 2100

ctgacccaca atcagtaaca tccatattgg gagatttttc aaaggatggt gaccctgctt 2160

gtatttattt accttggtat tttttcttgc atccttctgt gattcaaaaa agtaaaatgt 2220

ggctttctga aatgatggat aagagtctac atcttctaga aaaaatacat aaaggagtag 2280

ttaagctctg taaatgtgcc acgagctcca acacgaccat cgtagggtga agcccacgtt 2340

ttcttccatg gcctcaaagg ccctagaact tgcctacctt tctggcctta cctcctagct 2400

acttatccat ctcttgaact ttatactctt gtataaattt ctaactttca gaaaatgcca 2460

tactc gttt tggcaccaca catgtatatt tccccctggt acacttggaa gactcttatc 2520

catctgtgaa accctatgtt gtcatcactt ggtccatgaa atattacctg gccaatatcc 2580

caccatcacc tcaaacccaa tcaccccctc ctctgtatgc tgtcacacct atattattaa 2640

acttatcaca ttgcattgta attacttcct gacctttgta tctactcttt tagtaactga 2700

tgtatatatc tgaaaggaga gattgtttca ttgtgcaatc aataaatgtt tgataaaata 2760

aagccc 2766

<210>2

<211>1065

<212>DNA

＜213〉mankind

<400>2

atgactgccg ccataagaag acagagagaa ctgagtatcc tcccaaaggt gacactggaa 60

gcaatgaaca ccacagtgat gcaaggcttc aacagatctg agcggtgccc cagagacact 120

cggatagtac agctggtatt cccagccctc tacacagtgg ttttcttgac cggcatcctg 180

ctgaatactt tggctctgtg ggtgtttgtt cacatcccca gctcctccac cttcatcatc 240

tacctcaaaa acactttggt ggccgacttg ataatgacac tcatgcttcc tttcaaaatc 300

ctctc gact cacacctggc accctggcag ctcagagctt ttgtgtgtcg tttttcttcg 360

gtgatatttt atgagaccat gtatgtgggc atcgtgctgt tagggctcat agcctttgac 420

agattcctca agatcatcag acctttgaga aatatttttc taaaaaaacc tgtttttgca 480

aaaacggtct caatcttcat ctggttcttt ttgttcttca tctccctgcc aaatatgatc 540

ttgagcaaca aggaagcaac accatcgtct gtgaaaaagt gtgcttcctt aaaggggcct 600

ctggggctga aatggcatca aatggtaaat aacatatgcc agtttatttt ctggactgtt 660

tttatcctaa tgcttgtgtt ttatgtggtt attgcaaaaa aagtatatga ttcttataga 720

aagtccaaaa gtaaggacag aaaaaacaac aaaaagctgg aaggcaaagt atttgttgtc 780

gtggctgtct tctttgtgtg ttttgctcca tttcattttg ccagagttcc atatactcac 840

agtcaaacca acaataagac tgactgtaga ctgcaaaatc aactgtttat tgctaaagaa 900

acaactctct ttttggcagc aactaacatt tgtatggatc ccttaatata catattctta 960

tgtaaaaaat tcacagaaaa gctaccatgt atgcaaggga gaaagaccac agcatcaagc 1020

caagaaaatc atagcagtca gacagacaac ataaccttag gctga 1065

<210>3

<211>354

<212>PRT

＜213〉mankind

<400>3

Met Thr Ala Ala Ile Arg Arg Gln Arg Glu Leu Ser Ile Leu Pro Lys

1 5 10 15

Val Thr Leu Glu Ala Met Asn Thr Thr Val Met Gln Gly Phe Asn Arg

20 25 30

Ser Glu Arg Cys Pro Arg Asp Thr Arg Ile Val Gln Leu Val Phe Pro

35 40 45

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

50 55 60

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

65 70 75 80

Tyr Leu Lys Asn Thr Leu Val Ala Asp Leu Ile Met Thr Leu Met Leu

85 90 95

Pro Phe Lys Ile Leu Ser Asp Ser His Leu Ala Pro Trp Gln Leu Arg

100 105 110

Ala Phe Val Cys Arg Phe Ser Ser Val Ile Phe Tyr Glu Thr Met Tyr

115 120 125

Val Gly Ile Val Leu Leu Gly Leu Ile Ala Phe Asp Arg Phe Leu Lys

130 135 140

Ile Ile Arg Pro Leu Arg Asn Ile Phe Leu Lys Lys Pro Val Phe Ala

145 150 155 160

Lys Thr Val Ser Ile Phe Ile Trp Phe Phe Leu Phe Phe Ile Ser Leu

165 170 175

Pro Asn Met Ile Leu Ser Asn Lys Glu Ala Thr Pro Ser Ser Val Lys

180 185 190

Lys Cys Ala Ser Leu Lys Gly Pro Leu Gly Leu Lys Trp His Gln Met

195 200 205

Val Asn Asn Ile Cys Gln Phe Ile Phe Trp Thr Val Phe Ile Leu Met

210 215 220

Leu Val Phe Tyr Val Val Ile Ala Lys Lys Val Tyr Asp Ser Tyr Arg

225 230 235 240

Lys Ser Lys Ser Lys Asp Arg Lys Asn Asn Lys Lys Leu Glu Gly Lys

245 250 255

Val Phe Val Val Val Ala Val Phe Phe Val Cys Phe Ala Pro Phe His

260 265 270

Phe Ala Arg Val Pro Tyr Thr His Ser Gln Thr Asn Asn Lys Thr Asp

275 280 285

Cys Arg Leu Gln Asn Gln Leu Phe Ile Ala Lys Glu Thr Thr Leu Phe

290 295 300

Leu Ala Ala Thr Asn Ile Cys Met Asp Pro Leu Ile Tyr Ile Phe Leu

305 310 315 320

Cys Lys Lys Phe Thr Glu Lys Leu Pro Cys Met Gln Gly Arg Lys Thr

325 330 335

Thr Ala Ser Ser Gln Glu Asn His Ser Ser Gln Thr Asp Asn Ile Thr

340 345 350

Leu Gly

<210>4

<211>1014

<212>DNA

＜213〉house mouse (Mus musculus)

<400>4

atgctcggga caatcaacac cactgggatg cagggcttca acaagtctga gcggtgcccc 60

agggacactc ggatgacaca gctgctgttc ccggttctct atactgtggt cttcctggca 120

ggcatcctgc tgaacaccgt ggccctctgg gtgttcgtcc acatccccag caattccacc 180

tttatcgtct acctcaagaa cactctggtg gcagacttga taatggcact catgctgcct 240

ttcaaaatcc tttccgactc acaccttgcg ccctggcagc tccgaggatt tgtgtgcacg 300

ctctcctccg tggtcttcta tgagacgatg tatgtgggta tcatgatgct gggcctcatc 360

gctttcgaca ggttcctcaa gatcatcatg ccgttcagga aaacctttgt caaaaagacg 420

gctttcgcaa aaacagtctc catttccgtc tggtccctga tgttcttcat ctccctgcca 480

aacatgatct tgaacaagga ggcaacgcca tcatccgtga agaagtgtgc atctttgaag 540

agtccccttg ggctgtggtg gcatcaggtg gtcagtcaca cctgccagtt cattttctgg 600

gctgtgttta ttctgatgct tctgttttat gcggtgatta ccaaaaaggt gtacaactcc 660

tataggaagt ttaggagtaa ggacagcagg cacaagcggc tggaggtgaa ggtatttatc 720

gtcatggctg tcttctttgt ctgctttgcc ccactgcatt ttgtcagaat accatacact 780

tacagtcaaa ccaccaataa gactgactgt aggttagaaa accagctgtt tattgctaaa 840

gaagcaactc tctttctggc aacaactaac atttgtatgg accccttaat atacataatt 900

ttatgcaaga agttcacaca aaaggtgcca tgtgtgagat ggggaaaggc aagaacagca 960

ggatcaagcg aagaccacca cagtagtcag acagacaaca tcaccctagc ctga 1014

<210>5

<211>338

<212>PRT

＜213〉house mouse

<400>5

Met Leu Gly Thr Ile Asn Thr Thr Gly Met Gln Gly Phe Asn Lys Ser

1 5 10 15

Glu Arg Cys Pro Arg Asp Thr Arg Met Thr Gln Leu Leu Phe Pro Val

20 25 30

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

35 40 45

Leu Trp Val Phe Val His Ile Pro Ser Asn Ser Thr Phe Ile Val Tyr

50 55 60

Leu Lys Asn Thr Leu Val Ala Asp Leu Ile Met Ala Leu Met Leu Pro

65 70 75 80

Phe Lys Ile Leu Ser Asp Ser His Leu Ala Pro Trp Gln Leu Arg Gly

85 90 95

Phe Val Cys Thr Leu Ser Ser Val Val Phe Tyr Glu Thr Met Tyr Val

100 105 110

Gly Ile Met Met Leu Gly Leu Ile Ala Phe Asp Arg Phe Leu Lys Ile

115 120 125

Ile Met Pro Phe Arg Lys Thr Phe Val Lys Lys Thr Ala Phe Ala Lys

130 135 140

Thr Val Ser Ile Ser Val Trp Ser Leu Met Phe Phe Ile Ser Leu Pro

145 150 155 160

Asn Met Ile Leu Asn Lys Glu Ala Thr Pro Ser Ser Val Lys Lys Cys

165 170 175

Ala Ser Leu Lys Ser Pro Leu Gly Leu Trp Trp His Gln Val Val Ser

180 185 190

His Thr Cys Gln Phe Ile Phe Trp Ala Val Phe Ile Leu Met Leu Leu

195 200 205

Phe Tyr Ala Val Ile Thr Lys Lys Val Tyr Asn Ser Tyr Arg Lys Phe

210 215 220

Arg Ser Lys Asp Ser Arg His Lys Arg Leu Glu Val Lys Val Phe Ile

225 230 235 240

Val Met Ala Val Phe Phe Val Cys Phe Ala Pro Leu His Phe Val Arg

245 250 255

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

260 265 270

Glu Asn Gln Leu Phe Ile Ala Lys Glu Ala Thr Leu Phe Leu Ala Thr

275 280 285

Thr Asn Ile Cys Met Asp Pro Leu Ile Tyr Ile Ile Leu Cys Lys Lys

290 295 300

Phe Thr Gln Lys Val Pro Cys Val Arg Trp Gly Lys Ala Arg Thr Ala

305 310 315 320

Gly Ser Ser Glu Asp His His Ser Ser Gln Thr Asp Asn Ile Thr Leu

325 330 335

Ala Glx

<210>6

<211>2717

<212>DNA

＜213〉mankind

<400>6

tatgtttatt ggtaacaggt gacactggaa gcaatgaaca ccacagtgat gcaaggcttc 60

aacagatctg agcggtgccc cagagacact cggatagtac agctggtatt cccagccctc 120

tacacagtgg ttttcttgac cggcatcctg ctgaatactt tggctctgtg ggtgtttgtt 180

cacatcccca gctcctccac cttcatcatc tacctcaaaa acactttggt ggccgacttg 240

ataatgacac tcatgcttcc tttcaaaatc ctctctgact cacacctggc accctggcag 300

ctcagagctt ttgtgtgtcg tttttcttcg gtgatatttt atgagaccat gtatgtgggc 360

atcgtgctgt tagggctcat agcctttgac agattcctca agatcatcag acctttgaga 420

aatatttttc taaaaaaacc tgtttttgca aaaacggtct caatcttcat ctggttcttt 480

ttgttcttca tctccctgcc aaatatgatc ttgagcaaca aggaagcaac accatcgtct 540

gtgaaaaagt gtgcttcctt aaaggggcct ctggggctga aatggcatca aatggtaaat 600

aacatatgcc agtttatttt ctggactgtt tttatcctaa tgcttgtgtt ttatgtggtt 660

attgcaaaaa aagtatatga ttcttataga aagtccaaaa gtaaggacag aaaaaacaac 720

aaaaagctgg aaggcaaagt atttgttgtc gtggctgtct tctttgtgtg ttttgctcca 780

tttcattttg ccagagttcc atatactcac agtcaaacca acaataagac tgactgtaga 840

ctgcaaaatc aactgtttat tgctaaagaa acaactctct ttttggcagc aactaacatt 900

tgtatggatc ccttaatata catattctta tgtaaaaaat tcacagaaaa gctaccatgt 960

atgcaaggga gaaagaccac agcatcaagc caagaaaatc atagcagtca gacagacaac 1020

ataaccttag gctgacaact gtacataggg ttaacttcta tttattgatg agacttccgt 1080

agataatgtg gaaatcaaat ttaaccaaga aaaaaagatt ggaacaaatg ctctcttaca 1140

ttttattatc ctggtgtaca gaaaagatta tataaaattt aaatccacat agatctattc 1200

ataagctgaa tgaaccatta ctaagagaat gcaacaggat acaaatggcc actagaggtc 1260

attatttctt tctttctttt tttttttttt aatttcaaga gcatttcact ttaacatttt 1320

ggaaaagact aaggagaaac gtatatccct acaaacctcc cctccaaaca ccttctcaca 1380

ttcttttcca caattcacat aacactactg cttttgtgcc ccttaaatgt agatatgtgc 1440

tgaaagaaaa aaaaaacgcc caactcttga agtccattgc tgaaaactgc agccaggggt 1500

tgaaagggat gcagacttga agagtctgag gaactgaagt gggtcagcaa gacctctgaa 1560

atcctgggta aaggattttc tccttacaat tacaaacagc ctctttcaca ttacaataat 1620

ataccatagg aggcacaagc accattatta agccactttg cttacacctt aagtgtgtac 1680

aattcaagtg tgagaatgct gtgttaacta ttctttggaa ttctccttct gtccagcaaa 1740

tactctaatg atggttaaac atggcaccta ctcagcaatg ccttcctgga ccacaacccc 1800

tatccccctg ccccaccctc ctcattaaaa acaaatactt ctactgtttg ggtgtgtgat 1860

agggttctca atgcagatct cccttttcta gttagctata ttcttgactg catccgctaa 1920

aaatgttaaa gcttcttgag agacagacat gccagatttt cttggtatct cccataatac 1980

gacctacagt ccatggtcta cagatgtttt aaatagaatt gctattctcg atacatacaa 2040

agacgtaatt gctgacccac aatcagtaac atccatattg ggagattttt caaaggatgg 2100

tgaccctgct tgtatttatt taccttggta ttttttcttg catccttctg tgattcaaaa 2160

aagtaaaatg tggctttctg aaatgatgga taagagtcta catcttctag aaaaaataca 2220

taaaggagta gttaagctct gtaaatgtgc cacgagctcc aacacgacca tcgtagggtg 2280

aagcccacgt tttcttccat ggcctcaaag gccctagaac ttgcctacct ttctggcctt 2340

acctcctagc tacttatcca tctcttgaac tttatactct tgtataaatt tctaactttc 2400

agaaaatgcc atactctgtt ttggcaccac acatgtatat ttccccctgg tacacttgga 2460

agactcttat ccatctgtga aaccctatgt tgtcatcact tggtccatga aatattacct 2520

ggccaatatc ccaccatcac ctcaaaccca atcaccccct cctctgtatg ctgtcacacc 2580

tatattatta aacttatcac attgcattgt aattacttcc tgacctttgt atctactctt 2640

ttagtaactg atgtatatat ctgaaaggag agattgtttc attgtgcaat caataaatgt 2700

ttgataaaat aaagccc 2717

<210>7

<211>333

<212>PRT

＜213〉mankind

<400>7

Met Asn Thr Thr Val Met Gln Gly Phe Asn Arg Ser Glu Arg Cys Pro

1 5 10 15

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

20 25 30

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

35 40 45

Val His Ile Pro Ser Ser Ser Thr Phe Ile Ile Tyr Leu Lys Asn Thr

50 55 60

Leu Val Ala Asp Leu Ile Met Thr Leu Met Leu Pro Phe Lys Ile Leu

65 70 75 80

Ser Asp Ser His Leu Ala Pro Trp Gln Leu Arg Ala Phe Val Cys Arg

85 90 95

Phe Ser Ser Val Ile Phe Tyr Glu Thr Met Tyr Val Gly Ile Val Leu

100 105 110

Leu Gly Leu Ile Ala Phe Asp Arg Phe Leu Lys Ile Ile Arg Pro Leu

115 120 125

Arg Asn Ile Phe Leu Lys Lys Pro Val Phe Ala Lys Thr Val Ser Ile

130 135 140

Phe Ile Trp Phe Phe Leu Phe Phe Ile Ser Leu Pro Asn Met Ile Leu

145 150 155 160

Ser Asn Lys Glu Ala Thr Pro Ser Ser Val Lys Lys Cys Ala Ser Leu

165 170 175

Lys Gly Pro Leu Gly Leu Lys Trp His Gln Met Val Asn Asn Ile Cys

180 185 190

Gln Phe Ile Phe Trp Thr Val Phe Ile Leu Met Leu Val Phe Tyr Val

195 200 205

Val Ile Ala Lys Lys Val Tyr Asp Ser Tyr Arg Lys Ser Lys Ser Lys

210 215 220

Asp Arg Lys Asn Asn Lys Lys Leu Glu Gly Lys Val Phe Val Val Val

225 230 235 240

Ala Val Phe Phe Val Cys Phe Ala Pro Phe His Phe Ala Arg Val Pro

245 250 255

Tyr Thr His Ser Gln Thr Asn Asn Lys Thr Asp Cys Arg Leu Gln Asn

260 265 270

Gln Leu Phe Ile Ala Lys Glu Thr Thr Leu Phe Leu Ala Ala Thr Asn

275 280 285

Ile Cys Met Asp Pro Leu Ile Tyr Ile Phe Leu Cys Lys Lys Phe Thr

290 295 300

Glu Lys Leu Pro Cys Met Gln Gly Arg Lys Thr Thr Ala Ser Ser Gln

305 310 315 320

Glu Asn His Ser Ser Gln Thr Asp Asn Ile Thr Leu Gly

325 330

<210>8

<211>27

<212>DNA

＜213〉artificial

<220>

＜223〉Oligonucleolide primers

<400>8

tatacatatg ttcagcagta ccaactc 27

<210>9

<211>27

<212>DNA

＜213〉artificial

<220>

＜223〉Oligonucleolide primers

<400>9

acaccagt gtatagatagca agaagtc 27

<210>10

<211>36

<212>DNA

＜213〉artificial

<220>

＜223〉Oligonucleolide primers

<400>10

cccgtcgaca tgctttcttt tatgacaaaa tccttg 36

<210>11

<211>36

<212>DNA

＜213〉artificial

<220>

＜223〉Oligonucleolide primers

<400>11

aaagcggccg cgaacagcag ctgtgtcatc cgagtg 36

<210>12

<211>38

<212>DNA

＜213〉artificial

<220>

＜223〉Oligonucleolide primers

<400>12

aaaggcgcgc caggcaagaa cagcaggatc aagcgaag 38

<210>13

<211>35

<212>DNA

＜213〉artificial

<220>

＜223〉Oligonucleolide primers

<400>13

aaacaattgt ggcttctgag gctatggaaa gagag 35

<210>14

<211>27

<212>DNA

＜213〉artificial

<220>

＜223〉Oligonucleolide primers

<400>14

atatggcaca tttggtccgc actgcac 27

<210>15

<211>27

<212>DNA

＜213〉artificial

<220>

＜223〉Oligonucleolide primers

<400>15

gatgaggaat gatgtcacac agatgag 27

<210>16

<211>27

<212>DNA

＜213〉artificial

<220>

＜223〉Oligonucleolide primers

<400>16

aaggtcaaga ttagcaagtg attccag 27

<210>17

<211>27

<212>DNA

＜213〉artificial

<220>

＜223〉Oligonucleolide primers

<400>17

ataccataca cttacagtca aaccacc 27

<210>18

<211>27

<212>DNA

＜213〉artificial

<220>

＜223〉Oligonucleolide primers

<400>18

ggtcttcgct tgatcctgct gttcttg 27

<210>19

<211>27

<212>DNA

＜213〉artificial

<220>

＜223〉Oligonucleolide primers

<400>19

ttggctaccc gtgatattgc tgaagag 27

<210>20

<211>27

<212>DNA

＜213〉artificial

<220>

＜223〉Oligonucleolide primers

<400>20

gtcgtgaccc atggcgatgc ctgcttg 27

<210>21

<211>13800

<212>DNA

＜213〉artificial

<220>

＜223〉knock out the plasmid construction thing

<400>21

ttacaaatat gttaaaacaa acatacaaga gaacctgaat acatatgttc agcagtaaca 60

actcaaaaag tcaaacaaat taaatgtcca tccgtaacag aatgtttata caattttgtt 120

tgtatgttct cttggactta tgtatacaag tcgtcattgt tgagtttttc agtttgttta 180

atttacaggt aggcattgtc cttaaccaaa atatgggaaa agttgtgttt atcattatga 240

tcagtgtcta tgtattaaca tagactaaga caaaaattaa cacagactaa aataagcatg 300

gaattggttt tatacccttt tcaacacaaa tagtaatact agtcacagat acataattgt 360

atctgattct gtttttaatt gtgtctgatt ttattcgtac aacacttaca cactagtata 420

aatataaatg acttcttgct atctatacac tggtgtttag agttttcaaa tagttgtttt 480

gtgtgtgtgt atgttttgtg ttgtgaatgt gtgatcatat ttatatttac tgaagaacga 540

tagatatgtg accacaaatc tcaaaagttt atcaacaaaa cacacacaca tacaaaacac 600

tgtgtgtgtg tgtgtgtgtg tgtatgtgtg tgtgtgtgca tgtccccttg tgagtgtggg 660

cctgtgtaca ctatcacaca catataggtg tcaaaggaca acacacacac acacacacac 720

acatacacac acacacacgt acaggggaac actcacaccc ggacacatgt gatagtgtgt 780

gtatatccac agtttcctgt acctcaggtg tcagacatca tttgtcattt tgtttgagat 840

agagtctcct ttttactgct gcatatacca gaccaaccac tctgcatgct tctaaggaat 900

tggagtccac agtctgtagt aaacagtaaa acaaactcta tctcagagga aaaatgacga 960

cgtatatggt ctggttggtg agacgtacga agattcctta tctgtttcca tatccccaat 1020

ctcgcaatac gaatgacaga attacatata cacattactg tgtctacttc tatatgggct 1080

caggagatct aagtttattc agacaaaggt ataggggtta gagcgttatg cttactgtct 1140

taatgtatat gtgtaatgac acagatgaag atatacccga gtcctctaga ttcaaataag 1200

ttccatgctt gtgtggcaag tgctttacct actgcgtcat ctcctatgta tttcatcttt 1260

aaaacatatg ctttctttta tgacaaaatc cttgtaacta aaggtacgaa cacaccgttc 1320

acgaaatgga tgacgcagta gaggatacat aaagtagaaa ttttgtatac gaaagaaaat 1380

actgttttag gaacattgat aatattaagc atttcaagta ttgctgtgaa tatattgcct 1440

gtttctgaag agatttttct aatactgatt ttcacttcag gacatctgct tgtaaactac 1500

ttataattcg taaagttcat aacgacactt atataacgga caaagacttc tctaaaaaga 1560

ttatgactaa aagtgaagtc ctgtagacga acatttgatg atagtgtatt taactcatta 1620

tcacttttgg ttaccctaat tggaaagttt taaaaaattc acatgctcta aggaaagtac 1680

ctcagtagat ttcagagtaa tatcacataa attgagtaat agtgaaaacc aatgggatta 1740

acctttcaaa attttttaag tgtacgagat tcctttcatg gagtcatcta aagtctcatt 1800

ataagagttc tctcctagga atatttgagt tcccacttca acttacatcc ttagtgaaaa 1860

tgaaagcaaa catctcaaca tttctaatgt tattatatga tattctcaag agaggatcct 1920

tataaactca agggtgaagt tgaatgtagg aatcactttt actttcgttt gtagagttgt 1980

aaagattaca ataatatact tgcatgttaa ctacatcacc agaagtccct ttgctctttg 2040

ccttgatcca gctcaggaat cctggaggtc tagcaaagga agtaggtgta ggcaacttcc 2100

acgtacaatt gatgtagtgg tcttcaggga aacgagaaac ggaactaggt cgagtcctta 2160

ggacctccag atcgtttcct tcatccacat ccgttgaagg attacagacc agtttgtccc 2220

atctgaccat actggttgga caatttacaa atttaacctt agacctgagt gtgtaccaga 2280

cagaactgag tgtccgttca taatgtctgg tcaaacaggg tagactggta tgaccaacct 2340

gttaaatgtt taaattggaa tctggactca cacatggtct gtcttgactc acaggcaagt 2400

gcttcttttt cctgtatcaa tcattgactc ttggcataag gacttcagga tgaagtgaac 2460

cactccagct gctctctcag gggtgtggtg gggttgggac cgaagaaaaa ggacatagtt 2520

agtaactgag aaccgtattc ctgaagtcct acttcacttg gtgaggtcga cgagagagtc 2580

cccacaccac cccaaccctg aaggcaggct ctaagtgcaa attctagggc ccagtggtaa 2640

gttagtggtg gtctctatta ccactatttt gggaaggtgc ttaatttctt cattttgatt 2700

ttccgtccga gattcacgtt taagatcccg ggtcaccatt caatcaccac cagagataat 2760

ggtgataaaa cccttccacg aattaaagaa gtaaaactaa ttcacatcta aaataagact 2820

ggactatgtt gttattgtaa gggctgtgat aacatagtta cctataatac catagctatt 2880

tttattttat tactttgagt aagtgtagat tttattctga cctgatacaa caataacatt 2940

cccgacacta ttgtatcaat ggatattatg gtatcgataa aaataaaata atgaaactca 3000

ctattcttag tgcataaaag aattccagtc tttaatttat tccagtcttt aaattattca 3060

gcaggctcct aaacactgaa acttttcaat gcatggggtt gataagaatc acgtattttc 3120

ttaaggtcag aaattaaata aggtcagaaa tttaataagt cgtccgagga tttgtgactt 3180

tgaaaagtta cgtaccccaa tttttgtttg tttgtttgtt tgtttgtttt tgtttgtttt 3240

ttgtttttta gttcttcctt cccggccttc gtgaactaat aagcccacag tattcctatt 3300

aaaaacaaac aaacaaacaa acaaacaaaa acaaacaaaa aacaaaaaat caagaaggaa 3360

gggccggaag cacttgatta ttcgggtgtc ataaggataa ttctttttca ttgactgagg 3420

aagctcgcat gacatcgtcc atgacaggcc tcactgtgag catatgatgg acgttctttt 3480

acccctaact atatgaaaag aagaaaaagt aactgactcc ttcgagcgta ctgtagcagg 3540

tactgtccgg agtgacactc gtatactacc tgcaagaaaa tggggattga tatacttttc 3600

gacactgttg tgtatttcat cataaaaagg gggcaagtat ttcagagtga gtataaataa 3660

cttcccaaat gaggggaaca aaaagcgaca ggtggacaag ctgtgacaac acataaagta 3720

gtatttttcc cccgttcata aagtctcact catatttatt gaagggttta ctccccttgt 3780

ttttcgctgt ccacctgttc cccttggagc tacggcccgc agcttggaga tggcacttcc 3840

acacaggcct agggagcagc gtgcagagag gcccttccaa gaggaacagg ctttccaaca 3900

gggaacctcg atgccgggcg tcgaacctct accgtgaagg tgtgtccgga tccctcgtcg 3960

cacgtctctc cgggaaggtt ctccttgtcc gaaaggttgt aatatcagct aaggaactta 4020

ccaaggggag ctctcattta acaagtgtgt agtagttatg aagatgactc agctagagaa 4080

gaccaaccat gaccatgcag ttatagtcga ttccttgaat ggttcccctc gagagtaaat 4140

tgttcacaca tcatcaatac ttctactgag tcgatctctt ctggttggta ctggtacgtc 4200

agctcaatcc atgaaaccca catgttggaa agatataact aacccactga aggaaaacac 4260

acacacacat acacacacac tcacacactc acaataaata tcgagttagg tactttgggt 4320

gtacaacctt tctatattga ttgggtgact tccttttgtg tgtgtgtgta tgtgtgtgtg 4380

agtgtgtgag tgttatttat tgtatttcac aaccaaaaga atgtaccttt gtgaccctag 4440

aaatgtccat tgctgatcag ttcctctttt caaagtttcc cacatttggc attttgaaat 4500

acataaagtg ttggttttct tacatggaaa cactgggatc tttacaggta acgactagtc 4560

aaggagaaaa gtttcaaagg gtgtaaaccg taaaacttta ttgaccaaaa tgtgatttct 4620

acttctcata ttatgaaatt atcctgtaca gaatttccct tgtttaaagt aaaaatttcc 4680

agaccttcaa tgttatattg aactggtttt acactaaaga tgaagagtat aatactttaa 4740

taggacatgt cttaaaggga acaaatttca tttttaaagg tctggaagtt acaatataac 4800

gattgggggc tattgagaaa tttaaaattg actgtgttta tggttaaaat taacaataaa 4860

cctactttaa aatattgatt ttgctaggtt tttatcctct ctaacccccg ataactcttt 4920

aaattttaac tgacacaaat accaatttta attgttattt ggatgaaatt ttataactaa 4980

aacgatccaa aaataggaga tataaagcaa aaggatatat atttaagtta aggggcatta 5040

catcagtaaa tcctaacata ttttagccaa aaagaaaatt tttacaatgt gccatttatt 5100

atatttcgtt ttcctatata taaattcaat tccccgtaat gtagtcattt aggattgtat 5160

aaaatcggtt tttcttttaa aaatgttaca cggtaaataa ccctagagtt aaccaacgtt 5220

ccctggaaac acagtacatt tcattttaaa atgttcaaat gtggaatcaa tggattccaa 5280

attctcaacc aagtaatata gggatctcaa ttggttgcaa gggacctttg tgtcatgtaa 5340

agtaaaattt tacaagttta caccttagtt acctaaggtt taagagttgg ttcattatat 5400

ggaattacta gttagccaaa ccctgaagac ggtccatctc tacggtaagc gcgtggcaag 5460

aacggtgaag tgaaacttgc catagagaac cacacatcct ccttaatgat caatcggttt 5520

gggacttctg ccaggtagag atgccattcg cgcaccgttc ttgccacttc actttgaacg 5580

gtatctcttg gtgtgtagga ccccaggatc atctgatttc ctctttccct tcatctgtcg 5640

ggcagggaag cagactgtca cacattgtaa ctttctgcac actccctgag atttaaaacg 5700

ggggtcctag tagactaaag gagaaaggga agtagacagc ccgtcccttc gtctgacagt 5760

gtgtaacatt gaaagacgtg tgagggactc taaattttgc aaacactgta ccaatctgag 5820

gccagccctg attcaaaact tctctaagtc tcaagaatgg aggtggtttc ccaaaagggc 5880

ttcctaaaag tgccacactg tttgtgacat ggttagactc cggtcgggac taagttttga 5940

agagattcag agttcttacc tccaccaaag ggttttcccg aaggattttc acggtgtgac 6000

tagctccact caaactgggc aattcaagga acccaagcaa tgagcggctg gcatgtttag 6060

ctttcactga tgtgccctgg gtcggtccat cggtccccct atcgaggtga gtttgacccg 6120

ttaagttcct tgggttcgtt actcgccgac cgtacaaatc gaaagtgact acacgggacc 6180

cagccaggta gccaggggga cccccacact cctatgaagt agatgatgat ttcaaacttg 6240

ctacatgaga gaatgaggta tatagctctt aaaacatttg tttaaacata ataccgaata 6300

gggggtgtga ggatacttca tctactacta aagtttgaac gatgtactct cttactccat 6360

atatcgagaa ttttgtaaac aaatttgtat tatggcttat atgcagaggt ccaggaataa 6420

tagctaccac ttcttgggaa atgtgactca taatctgcca cagatctagg gatcaaaaga 6480

ctaacagcaa tgtctgaaga tacgtctcca ggtccttatt atcgatggtg aagaaccctt 6540

tacactgagt attagacggt gtctagatcc ctagttttct gattgtcgtt acagacttct 6600

aaggaaacaa agcaaaacaa acaagcaacc aacaaccaaa gcctagcctt agacatacaa 6660

acttcttgca cgcccaggct gtaagggaca gaagatgatt ttcctttgtt tcgttttgtt 6720

tgttcgttgg ttgttggttt cggatcggaa tctgtatgtt tgaagaacgt gcgggtccga 6780

cattccctgt cttctactaa tgattttcag atcaagtctt ctcctcctac ttgattgtaa 6840

tttcatacta ttttcttcat cattttaact cctactatta catatgaagt attcaataag 6900

actaaaagtc tagttcagaa gaggaggatg aactaacatt aaagtatgat aaaagaagta 6960

gtaaaattga ggatgataat gtatacttca taagttattc gtcgctgaat gtgaataata 7020

aaaagtaaaa cactgggcct agagaaatag ttcggcggtt aaagggattt tcctggcacc 7080

cactccaggc agctcacagt cagcgactta cacttattat ttttcatttt gtgacccgga 7140

tctctttatc aagccgccaa tttccctaaa aggaccgtgg gtgaggtccg tcgagtgtca 7200

caccttggac cccagctcca gggaatccaa cacttctggc ctctgagggc acctgaatgc 7260

atatgcgccc ttacacatat aatttaatat aataaaatag gtggaacctg gggtcgaggt 7320

cccttaggtt gtgaagaccg gagactcccg tggacttacg tatacgcggg aatgtgtata 7380

ttaaattata ttattttatc accacatgtt aagaatatgg cgtggtggcg tgcctgcaga 7440

gccggtgaca gcactggatt caatactcag aacacgcaac caataactac tggtaaatgc 7500

tggtgtacaa ttcttatacc gcaccaccgc acggacgtct cggccactgt cgtgacctaa 7560

gttatgagtc ttgtgcgttg gttattgatg accatttacg tccaaaggtc taaagacaaa 7620

gaggttctag aaaatgtggg tattacagaa gcccacaagg ggaaatgagg aaggaaaagc 7680

agtataagaa agcagaacct aggtttccag atttctgttt ctccaagatc ttttacaccc 7740

ataatgtctt cgggtgttcc cctttactcc ttccttttcg tcatattctt tcgtcttgga 7800

ttacttcaca agctattcgt gggttgagct agtaactgcc acaactcata cagctgagtc 7860

tcttccaaaa caaaggtcag tttaattttt cttatgaatt aatgaagtgt tcgataagca 7920

cccaactcga tcattgacgg tgttgagtat gtcgactcag agaaggtttt gtttccagtc 7980

aaattaaaaa gaatacttaa tgcttgctat aaaaggtaaa aggtggttaa attttctaga 8040

aatctaaata taatattctg taatcacggg ctgcaagcat aaggtacttc ataacgcatg 8100

acgaacgata ttttccattt tccaccaatt taaaagatct ttagatttat attataagac 8160

attagtgccc gacgttcgta ttccatgaag tattgcgtac acacactacc ctgtactctt 8220

acatcttaaa ttaggttctg tcaccgccgt gtagtgattg gtgcttgcta tttggacttc 8280

acaagggtag acattaacag tgtgtgatgg gacatgagaa tgtagaattt aatccaagac 8340

agtggcggca catcactaac cacgaacgat aaacctgaag tgttcccatc tgtaattgtc 8400

gactttataa aacaaaacta aactgaacaa aaaccaacca aacaacaaca gcaacaaaac 8460

taagctatgt gctcaaaaaa gctctagcca aagaatcaaa ctgaaatatt ttgttttgat 8520

ttgacttgtt tttggttggt ttgttgttgt cgttgttttg attcgataca cgagtttttt 8580

cgagatcggt ttcttagttt tccgctgaat gagttaaata gtaagtttgt ttttacaaga 8640

agtagttatc tgccaatcca caaggaccct ttgtttacct tcctttgtat gcctgcttat 8700

aggcgactta ctcaatttat cattcaaaca aaaatgttct tcatcaatag acggttaggt 8760

gttcctggga aacaaatgga aggaaacata cggacgaata gtgtaatctt tctctgtgtg 8820

catgtttatg tgtaatagct gatgctcggg acaatcaaca ccactgggat gcagggcttc 8880

aacaagtctg agcggtgccc cacattagaa agagacacac gtacaaatac acattatcga 8940

ctacgagccc tgttagttgt ggtgacccta cgtcccgaag ttgttcagac tcgccacggg 9000

cagggacact cggatgacac agctgctgtt cccggttctc tatactgtgg tcttcctggc 9060

aggcatcctg ctgaacaccg tggccctctg ggtgttcgtc gtccctgtga gcctactgtg 9120

tcgacgacaa gggccaagag atatgacacc agaaggaccg tccgtaggac gacttgtggc 9180

accgggagac ccacaagcag cacatcccca gcaattccac ctttatcgtc tacctcaaga 9240

acactctggt ggcagacttg ataatggcac tcatgctgcc tttcaaaatc ctttccgact 9300

gtgtaggggt cgttaaggtg gaaatagcag atggagttct tgtgagacca ccgtctgaac 9360

tattaccgtg agtacgacgg aaagttttag gaaaggctga cacaccttgc gccctggcag 9420

ctccgaggat ttgtgtgcac gctctcctcc gtggtcttct atgagacgat gtatgtgggt 9480

atcatgatgc tgggcctcat gtgtggaacg cgggaccgtc gaggctccta aacacacgtg 9540

cgagaggagg caccagaaga tactctgcta catacaccca tagtactacg acccggagta 9600

cgctttcgac aggttcctca agatcatcat gccgttcagg aaaacctttg tcaaaaagac 9660

ggctttcgca aaaacagtct ccatttccgt ctggtccctg gcgaaagctg tccaaggagt 9720

tctagtagta cggcaagtcc ttttggaaac agtttttctg ccgaaagcgt ttttgtcaga 9780

ggtaaaggca gaccagggac atgttcttca tctccctgcc aaacatgatc ttgaacaagg 9840

aggcaacgcc atcatccgtg aagaagtgtg catctttgaa gagtcccctt gggctgtggt 9900

tacaagaagt agagggacgg tttgtactag aacttgttcc tccgttgcgg tagtaggcac 9960

ttcttcacac gtagaaactt ctcaggggaa cccgacacca ggcatcaggt ggtcagtcac 10020

acctgccagt tcattttctg ggctgtgttt attctgatgc ttctgtttta tgcggtgatt 10080

accaaaaagg tgtacaactc ccgtagtcca ccagtcagtg tggacggtca agtaaaagac 10140

ccgacacaaa taagactacg aagacaaaat acgccactaa tggtttttcc acatgttgag 10200

ctataggaag tttaggagta aggacagcag gcacaagcgg ctggaggtga aggtatttat 10260

cgtcatggct gtcttctttg tctgctttgc cccactgcat gatatccttc aaatcctcat 10320

tcctgtcgtc cgtgttcgcc gacctccact tccataaata gcagtaccga cagaagaaac 10380

agacgaaacg gggtgacgta tttgtcagaa taccatacac ttacagtcaa accaccaata 10440

agactgactg taggttagaa aaccagctgt ttattgctaa agaagcaact ctctttctgg 10500

aaacagtctt atggtatgtg aatgtcagtt tggtggttat tctgactgac atccaatctt 10560

ttggtcgaca aataacgatt tcttcgttga gagaaagacc caacaactaa catttgtatg 10620

gaccccttaa tatacataat tttatgcaag aagttcacac aaaaggtgcc atgtgtgaga 10680

tggggaaagg caagaacagc gttgttgatt gtaaacatac ctggggaatt atatgtatta 10740

aaatacgttc ttcaagtgtg ttttccacgg tacacactct acccctttcc gttcttgtcg 10800

aggatcaagc gaagaccacc acagtagtca gacagacaac atcaccctag cctgaccact 10860

gtgtcccaca ggttaatttc acgcatggcc tcacgtctat tcctagttcg cttctggtgg 10920

tgtcatcagt ctgtctgttg tagtgggatc ggactggtga cacagggtgt ccaattaaag 10980

tgcgtaccgg agtgcagata ttatggatgg gatttcaaaa gatcatttat gtggagacct 11040

catttaagca ttacaggaaa aaagagggga acaaacagtt ccctacattt tattatccta 11100

aatacctacc ctaaagtttt ctagtaaata cacctctgga gtaaattcgt aatgtccttt 11160

tttctcccct tgtttgtcaa gggatgtaaa ataataggat gtgtatggaa aaacattatg 11220

cccattttaa ccacatagac gtatttataa gcaggataaa ttaagagacc atgtaataca 11280

gcaaatggcc actagatgtc cacatacctt tttgtaatac gggtaaaatt ggtgtatctg 11340

cataaatatt cgtcctattt aattctctgg tacattatgt cgtttaccgg tgatctacag 11400

accttttcaa gggcattcat gtactatgga aaaggttaat gggaaacagg tttgcctgaa 11460

aaatcttcct tctagttacc accccaccat ctcttcacac tggaaaagtt cccgtaagta 11520

catgatacct tttccaatta ccctttgtcc aaacggactt tttagaagga agatcaatgg 11580

tggggtggta gagaagtgtg atatattccc taaaacacca ggctggcttt tacagccttc 11640

agaatgctga cacttgtgaa cagaaaccaa ccaacttgca tatccagtgc ctgtgtggaa 11700

tatataaggg attttgtggt ccgaccgaaa atgtcggaag tcttacgact gtgaacactt 11760

gtctttggtt ggttgaacgt ataggtcacg gacacacctt aggctaaggt gggggctcaa 11820

agagatgcag tctgaggaac caaagtgggt tggtcaaaat aaccccaggc atctcaaaag 11880

atttcctcct tacaagtgca tccgattcca cccccgagtt tctctacgtc agactccttg 11940

gtttcaccca accagtttta ttggggtccg tagagttttc taaaggagga atgttcacgt 12000

aagggctgct cctacatcta aacagagcac cagaagagag gcacatgcaa caggcaaagc 12060

cagttcacag ccatgtgcaa tccagagagg ggaagtgttt ttcccgacga ggatgtagat 12120

ttgtctcgtg gtcttctctc cgtgtacgtt gtccgtttcg gtcaagtgtc ggtacacgtt 12180

aggtctctcc ccttcacaaa agtcagcaaa accttcctgg gcaacagcat ctgcatcctg 12240

ttggaaaata cttatttccc tcactgttta tctataaatt aggttccttg ctacacactg 12300

tcagtcgttt tggaaggacc cgttgtcgta gacgtaggac aaccttttat gaataaaggg 12360

agtgacaaat agatatttaa tccaaggaac gatgtgtgac tcttatatta actgctttca 12420

ttcttagcca catttcccaa aaacagggtt cgtaaaaaga cagcaaaatc acacattttt 12480

acaaaagaaa tggggtaagg agaatataat tgacgaaagt aagaatcggt gtaaagggtt 12540

tttgtcccaa gcatttttct gtcgttttag tgtgtaaaaa tgttttcttt accccattcc 12600

atatcctaga cgggatgttt gttgtacact atccttagtg catgtgagca aggggatggt 12660

tggcctggca ttagtaatat tcatgtggga agatttttca tataggatct gccctacaaa 12720

caacatgtga taggaatcac gtacactcgt tcccctacca accggaccgt aatcattata 12780

agtacaccct tctaaaaagt aagcctgatt cattttattt gggcctatct acttccctgg 12840

atctcattat gggtttaaga aaattaaaat atgtggctgg tgatgctggg ttttctggag 12900

ttcggactaa gtaaaataaa cccggataga tgaagggacc tagagtaata cccaaattct 12960

tttaatttta tacaccgacc actacgaccc aaaagacctc aacacgacga ctcttcccct 13020

aaccgcgcct gtaatgtgaa atcctggctt ctctctttcc atagcctcag aagccacaga 13080

ggcaagagaa cttccttgtc ttgtgctgct gagaagggga ttggcgcgga cattacactt 13140

taggaccgaa gagagaaagg tatcggagtc ttcggtgtct ccgttctctt gaaggaacag 13200

ttcctggaat cacttgctaa tcttgacctt tacaaactcc atttaatatg gcacatttgg 13260

tccgcactgc actgtacata gaaccttcca tctgacccac aaggacctta gtgaacgatt 13320

agaactggaa atgtttgagg taaattatac cgtgtaaacc aggcgtgacg tgacatgtat 13380

cttggaaggt agactgggtg tttgtaagtt cctattgatt agtgacaccc aaagttgctg 13440

tcccttactc caggtaacct tacctgccca acctcctccg agtcctccag cccagtcact 13500

aaacattcaa ggataactaa tcactgtggg tttcaacgac agggaatgag gtccattgga 13560

atggacgggt tggaggaggc tcaggaggtc gggtcagtga tcctcatctg tgtgacatca 13620

ttcctcatct acttatcgaa ttgatttagg ataatttgct aaatatgcac tgtgcaatta 13680

ataaattttg ctaaaacaaa aggagtagac acactgtagt aaggagtaga tgaatagctt 13740

aactaaatcc tattaaacga tttatacgtg acacgttaat tatttaaaac gattttgttt 13800

Claims (28)

1. identify and be suitable for treatment, prevent or alleviate the GPR86 relevant disease, the method of the molecule of inflammatory disease or pain particularly, this method comprises determining whether candidate molecules is the activator or the antagonist of GPR86 polypeptide, wherein said GPR86 polypeptide comprise amino acid sequence shown in SEQ ID NO:3, SEQ ID NO:5 or the SEQ ID NO:7, its fragment or with its at least 90% identical sequence.

According to the process of claim 1 wherein described GPR86 polypeptide by nucleotide sequence shown in SEQ ID NO:1, SEQ IDNO:2 or the SEQ ID NO:4 or with its at least 90% identical sequential coding.

3. according to the method for claim 1 or 2, comprise candidate molecules being exposed to the GPR86 polypeptide and determining that whether candidate molecules is in conjunction with the GPR86 polypeptide.

4. according to claim 1,2 or 3 method, wherein the cells contacting candidate compound of the G Protein G i coupling by making contained GPR86 acceptor and GPR86 preference and the level of determining GPCR sensitivity label thing such as cyclic adenosine monophosphate (cAMP) in the described cell whether since described contact to reduce identify activator.

5. according to claim 1,2 or 3 method, wherein the cells contacting candidate compound of the G Protein G i coupling by making contained GPR86 acceptor and GPR86 preference and the level of determining GPCR sensitivity label thing such as cyclic adenosine monophosphate (cAMP) in the described cell whether since described contact to improve identify antagonist.

6. according to claim 1,2 or 3 method, wherein by making contained GPR86 acceptor and the pungency G albumen that mixes such as G _{α 16}The cells contacting candidate compound of coupling also determines whether the level of GPCR sensitivity label thing such as cyclic adenosine monophosphate (cAMP) in the described cell identifies activator owing to described contact improves.

7. according to claim 1,2 or 3 method, wherein by making contained GPR86 acceptor and the pungency G albumen that mixes such as G _{α 16}The cells contacting candidate compound of coupling also determines whether the level of GPCR sensitivity label thing such as cyclic adenosine monophosphate (cAMP) in the described cell identifies antagonist owing to described contact reduces.

8. according to the method for claim 1 or 2, comprising: the transgenic nonhuman animal that (a) provides the function of wild type animal or endogenous GPR86 gene to be destroyed; (b) wild type or transgenic nonhuman animal are exposed to candidate molecules; And whether the biological parameter of (c) determining animal is owing to described contact changes.

9. according to the method for claim 8, wherein said biological parameter be selected to the reaction that stimulates, to the reaction of heat, to light reaction, immune response, inflammatory reaction, to the reaction of pain, preferably to the reaction of pain.

10. according to the method for claim 1 or 2, comprising: the cell that (a) provides the function of wild-type cell or endogenous GPR86 gene to be destroyed, preferably isolated cells from the transgenic nonhuman animal that the function of endogenous GPR86 gene is destroyed; (b) with cellular exposure in candidate molecules; And whether the biologic activity of (c) determining the GPR86 polypeptide is owing to described contact changes.

11. the transgenic nonhuman animal that the function of wild type animal or endogenous GPR86 gene is destroyed is used for the treatment of, prevents in evaluation or alleviates purposes in the method for the GPR86 polypeptide activator of GPR86 relevant disease, particularly inflammatory disease or pain or antagonist.

12. the transgenic nonhuman animal that the function of endogenous GPR86 gene is destroyed or its isolated cells or tissue are as the purposes of GPR86 relevant disease, particularly inflammatory disease or pain model.

13. according to each purposes or method of claim 8-12, wherein said transgenic nonhuman animal comprises the GPR86 gene that function is destroyed, and preferably comprises deletion in GPR86 gene or its part.

14. according to each purposes or method of claim 8-13, wherein compare with the wild type animal, transgenic nonhuman animal demonstrates variation in following each or multinomial phenotype: to the reaction that stimulates, to the reaction of heat, to light reaction, immune response, inflammatory reaction, to the reaction of pain, preferably to the reaction of pain.

15. according to each purposes or method of claim 8-14, wherein compare with the wild type animal, transgenic nonhuman animal demonstrates following at least one: (a) neurological susceptibility to pain changes, promptly the susceptibility to pain improves or reduces, (b) neurological susceptibility to inflammatory pain changes, and promptly the neurological susceptibility to inflammatory pain improves or reduces.

16. according to each purposes or method of claim 8-15, wherein said transgenic nonhuman animal is a rodent, preferred mouse.

17. identify the activator of GPR86 polypeptide or the method for antagonist, this method comprises to according to each wild type or the transgenic nonhuman animal variation of using candidate compound and measuring listed biological parameter in the claim 9 of claim 4-16.

18. comprise amino acid sequence shown in SEQ ID NO:3, SEQ ID NO:5 or the SEQ ID NO:7, its fragment or identify and be used for the treatment of, prevent its activator of GPR86 relevant disease, particularly inflammatory disease or pain or the purposes of antagonist with the GPR86 polypeptide of its at least 90% identical sequence.

19. comprise nucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:2 or the SEQ ID NO:4, its fragment or identify and be used for the treatment of, prevent its activator of GPR86 relevant disease, particularly inflammatory disease or pain or the purposes of antagonist with the GPR86 polynucleotide of its at least 90% identical sequence.

20. method according to any aforementioned claim, wherein said pain is selected from acute pain, chronic pain, dermatalgia, somatalgia, splanchnodynia, referred pain comprises myocardial ischemia, unreal painful and neuropathic pain (neuralgia), by damage, the pain that disease causes, headache, antimigraine, cancer pain, the pain that causes by neurological disorder such as Parkinson's disease, by backbone and operation on peripheral nerve, brain tumor, traumatic brain injury (TBI), trauma of spinal cord, chronic pain syndrome, the pain that chronic fatigue syndrome causes, neuralgia is such as trigeminal neuralgia, glossopharyngeal neuralgia, postherpetic neuralgia and causalgia, by lupus, sarcoidosis, archnoiditis, arthritis, the pain that rheumatic disease causes, periodicity pain, backache, back pain, arthralgia, stomachache, pectoralgia, pain of childbirth, muscle skeleton and disease of skin, head trauma and fibromyalgia.

21. method according to any aforementioned claim, wherein said inflammatory disease is selected from the inflammatory disorder, is preferably selected from inflammatory disease (rheumatoid arthritis for example, multiple sclerosis, guillain-Barre syndrome, Crohn's disease, ulcerative colitis, psoriasis, graft versus host disease(GVH disease), systemic lupus, lupus erythematosus or insulin-dependent diabetes), autoimmune disease (TSS for example, osteoarthritis, diabetes or inflammatory bowel disease), acute pain, chronic pain, neuropathic pain, contact dermatitis, atherosclerotic, glomerulonephritis, reperfusion injury, bone-resorbing disease, asthma, apoplexy, miocardial infarction, thermal burn, adult respiratory distress syndrome (ARDS) (ARDS), the multiple organ injury of wound secondary, skin disease with the acute inflammation composition, acute purulent meningitis, gangrenosum acne intestines colitis, with haemodialysis, infectious shock, the leucocyte removal art, the syndrome that the granulocyte blood transfusion is relevant, suck the acute or chronic inflammation of lung that causes by smog, mullerianosis, Behcet, uveitis, ankylosing spondylitis, pancreatitis, cancer, Lyme disease, ISR after skin is worn the chamber coronary angioplasty, Alzheimer's, traumatic arthritis, pyemia, chronic obstructive pulmonary disease, congestive heart failure, osteoporosis, cachexia, Parkinson's disease, periodontosis, gout, allergic disease, the age related macular degeneration, infect and cystic fibrosis.

22. by GPR86 activator or the antagonist of identifying according to the method or the purposes of any aforementioned claim.

23. the molecule according to claim 22 is used for the treatment of, prevents or alleviate the purposes of GPR86 relevant disease, particularly inflammatory disease or pain.

24.GPR86 the diagnostic kit of relevant disease, particularly inflammatory disease or pain or its neurological susceptibility, comprise following each or multinomial: GPR86 polypeptide or its part; Antibody at the GPR86 polypeptide; Maybe can the encode nucleic acid of above-mentioned substance.

25. treatment suffers from the method for the individuality of GPR86 relevant disease, particularly inflammatory disease or pain, this method comprises activity or the content that improves or reduce GPR86 polypeptide in the individuality.

26. according to the method for claim 25, this method comprises to individuality uses GPR86 polypeptide, GPR86 polypeptide activator or GPR86 antagonist.

27. the method for diagnosis GPR86 relevant disease, particularly inflammatory disease or pain, the method comprising the steps of: (a) detect and suffer from or suspect GPR86 polypeptide expression level or pattern in the animal that suffers from this type of disease; And (b) expression or pattern and intact animal are compared.

28. as mentioned above and with reference to the accompanying drawings 1-7 or method as shown in it or purposes basically.

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