CN101031799A - Gpr100 receptor used in application of diabetes mellitus and obesity regulation - Google Patents

Abstract

We describe a method of identifying a molecule suitable for the treatment, prophylaxis or alleviation of a Gpr100 associated disease, in particular diabetes and obesity, the method comprising determining whether a candidate molecule is an agonist or antagonist of Gpr100 polypeptide, in which the Gpr100 polypeptide comprises the amino acid sequence shown in SEQ ID NO. 3 or SEQ ID NO. 5, or a sequence which is at least 90% identical thereto.

Description

The purposes of GPR100 acceptor in diabetes and obesity adjusting

Technical field

The present invention relates to the new nucleic acid of identifying, by its encoded polypeptides with and generation and purposes.More specifically, nucleic acid of the present invention and polypeptide relate to g protein coupled receptor (GPCR), hereinafter referred to as " Gpr100GPCR ".The invention still further relates to the effect that suppresses or activate described nucleic acid and polypeptide.

Background technology

Determined many medically important biological processes by comprising G-albumen and/or second messenger, for example the protein mediation of the participation signal transduction pathway of cAMP (Lefkowitz, Nature, 1991,351:353-354).These protein are called the protein that participation has the approach of G-albumen or " PPG albumen ".Some examples of these protein comprise gpc receptor, as are used for those (Kobilka, B.K., et al., Proc.Natl Acad.Sci., USA, 1987,84:46-50 of adrenergic agent 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 driving 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 form of signal transduction, the activation that act as adenyl cyclase in the small cell of hormone combination.Enzyme relies on nucleotide, the existence of GTP by the activation of hormone.GTP also influences the combination of hormone.G albumen is connected to adenyl cyclase with hormone receptor.G albumen shows the GDP with the GTP exchange when being activated by hormone receptor.The form of carrying GTP is subsequently in conjunction with the adenyl cyclase that activates.GTP is hydrolyzed into GDP by G albumen autocatalysis, and G albumen is got back to its basic, inactive form.Therefore, G albumen provides double action, as intermediate signal is handover to effector molecules from acceptor, and as duration of clock control signal.

The membrane protein gene superfamily of g protein coupled receptor (GPCR) has been characterized by has seven membrane spaning domains of inferring.Think transmembrane spanning that the representative of this domain is outer by born of the same parents or the kytoplasm ring connects.G protein coupled receptor comprises various biologic activity acceptors, as hormone, virus, growth factor and neuroceptor.

G protein coupled receptor (also claiming the 7TM acceptor) has been characterized by and has comprised about 20 to 30 amino acid whose these seven conservative hydrophobic tracts (Stretch), connects at least eight and disperses hydrophilic loop.G protein coupled receptor family comprises in conjunction with the dopamine receptor that is used for the treatment of the psychosis medicine of mental disease and neurology illness.Other examples of this family member include but not limited to, calcitonin, adrenergic, Endothelin (endothelin), cAMP, adenosine, muscarine, acetylcholine, serotonin, histamine, fibrin ferment, kassinin kinin, follicle-stimulating hormone (FSH), opsin, endothelial differentiation gene-1, rhodopsin, deodorant and cytomegalovirus acceptor.

Most of g protein coupled receptors have single conserved cysteine residue in each of preceding two born of the same parents' outer shrouds of disulfide bond of stabilization function protein structure thought in formation.7 membrane-spanning domains are called TM1, TM2, TM3, TM4, TM5, TM6 and TM7.TM3 relates to signal transduction.

The phosphorylation of cysteine residues and fatization (palmitoylation or farnesylation) can influence the signal transduction of some g protein coupled receptors.Most of g protein coupled receptors comprise potential phosphorylation site in the 3rd kytoplasm ring and/or carboxyl terminal.For some g protein coupled receptors, as receptor,, the desensitization of the phosphorylation mediation acceptor by protein kinase A and/or specific receptor kinase.For some acceptors, think that the ligand-binding site point of g protein coupled receptor comprises the hydrophilic nest (socket) that is formed by several g protein coupled receptor membrane spaning domains, this nest is surrounded by the hydrophobic residue of g protein coupled receptor.Think that the water-wet side of each g protein coupled receptor transbilayer helix is towards inner and form the polar ligand binding site.TM3 is associated with several g protein coupled receptors as the TM3 asparagicacid residue because of having the ligand-binding site point.TM5 serine, TM6 asparagine and TM6 or TM7 phenylalanine or tyrosine also participate in the part combination.

G protein coupled receptor can by be coupled in the heterotrimeric G-albuminous cell different desmoenzymes, ion channel and transport protein (referring to, Johnson et al., Endoc.Rev., 1989,10:317-331).Different G protein alpha-subunits preferentially stimulates specific effector molecules to adjust different biological function in the cell.Determined that the phosphoric acid of g protein coupled receptor kytoplasm residue turns to the important mechanisms of the G albumen coupling of regulating some g protein coupled receptors.G protein coupled receptor is found at many positions in mammalian hosts.In in the past 15 years, 7 of similar 350 kinds of targets are striden the therapeutic agent of film (7 TM) acceptor and are successfully introduced market.

Therefore, g protein coupled receptor has the history of conduct treatment target definite, that confirm.Obviously, needing to identify and characterize can be in prevention, other acceptor that works in improvement or arbor press dysfunction or the disease, described dysfunction or disease include, without being limited to, obesity, comprise the prevention of obesity or weight increase, appetite suppresses, the lipid metabolism illness, comprise hyperlipidemia, dyslipidemia (dyslipidemia) and hypertriglyceridemia, depression and anxiety, diabetes and associated conditions include but not limited to: type i diabetes, type ii diabetes, impaired glucose tolerance, insulin resistance syndrome, the X syndrome, hyperglycemia, acute pancreas adenositis, angiocardiopathy, hypertension, cardiomegaly and hypercholesterolemia.

Summary

Evaluation is suitable for treatment, prevents or alleviates the Gpr100 relevant disease, especially the method for the molecule of diabetes and obesity, described method comprises determines whether candidate molecules is the activator or the antagonist of Gpr100 polypeptide, wherein the Gpr100 polypeptide comprises the amino acid sequence shown in SEQ ID NO.3 or the SEQ ID NO:5, or with its at least 90% same sequence.

Preferably, the Gpr100 polypeptide by the nucleotide sequence shown in SEQ ID No.1, SEQ ID No.2 or the SEQ ID NO:4 or with its at least 90% same sequential coding.

Preferably, described method comprises candidate molecules contacted with the Gpr100 polypeptide, and detects the variation as described contact result's intracellular Ca2+ level.

Preferably, this method comprises makes non-human animal or its part, preferred cell, tissue or organ contact candidate molecules and determine whether change as the biological parameter of the described animal of described contact result.

Preferably, described biological parameter is selected from down the group parameter: serum level of glucose, body weight, hyperglycemic factor level, fatty percent (fat percentage).

According to a second aspect of the invention, provide transgenic nonhuman animal with the destroyed endogenous Gpr100 of function or its isolated cells or tissue to regulate or the Gpr100 relevant disease, the purposes of preferred obesity or diabetes model as glucose.

Preferably, described transgenic nonhuman animal comprises the destroyed Gpr100 gene of function, preferably includes the disappearance of Gpr100 gene or its part.

Preferably, described transgenic nonhuman animal is compared with the wild type animal and presented change any or multiple in the following phenotype: serum level of glucose reduction, weight increase, fatty percent are higher.

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

According to a third aspect of the present invention, provide and comprise the amino acid sequence shown in SEQ ID NO.3 or the SEQ ID NO:5, or be used for identifying treatment, prevention Gpr100 relevant disease, preferred obesity or the activator of diabetes or the purposes of its antagonist with the Gpr100 polypeptide of its at least 90% same sequence.

As the 4th aspect of the present invention, provide and comprise the nucleotide sequence shown in SEQ ID NO.1, SEQ ID No.2 or the SEQID NO:4, or be used for identifying treatment, prevention Gpr100 relevant disease, preferred obesity or the activator of diabetes or the purposes of its antagonist with the Gpr100 polynucleotide of its at least 90% same sequence.

According to a fifth aspect of the present invention, provide non-human animal or its part, preferred cell, tissue or organ are being identified treatment, preventing or are alleviating the Gpr100 relevant disease, the purposes in the activator of preferred diabetes or obesity or the method for antagonist.

The present invention provides aspect the 6th activator or the antagonist identified by the method for aforementioned any claim or purposes to be used for the treatment of, to prevent or alleviates the Gpr100 relevant disease, the purposes of preferred obesity or diabetes.

The 7th aspect of the present invention provides by regulating and control to comprise in the individuality amino acid sequence shown in SEQ ID NO.3 or the SEQ IDNO:5 or regulating and control the method for the adjusting of glucose in the individuality, fat metabolism or weight increase with the Gpr100 polypeptide active of its 90% same sequence at least.

Preferably, described method comprises that activator or antagonist with Gpr100 are administered to individuality.

According to an eighth aspect of the present invention, the method that provides treatment to suffer from the individuality of Gpr100 relevant disease, this method comprise the active or amount that improves or reduce Gpr100 polypeptide in the individuality.

Preferably, described method comprises the activator of Gpr100 polypeptide, Gpr100 polypeptide or the antagonist of Gpr100 is administered to described individuality.

According to a ninth aspect of the present invention, provide the diagnostic method of Gpr100 relevant disease, described method comprises step: (a) detect suffer from or the doubtful animal that suffers from described disease in Gpr100 polypeptide expression level or pattern; (b) comparing with described expression or pattern and intact animal.

According to a tenth aspect of the present invention, provide the diagnostic method of Gpr100 relevant disease, described method comprises and detects the change of biological parameter as mentioned above in the doubtful individuality of suffering from described disease.

As the 11 aspect of the present invention, the relevant disease to Gpr100 is provided, the diagnostic kit of the neurological susceptibility of preferred obesity or diabetes, described kit comprise in following any or multiple: Gpr100 polypeptide or its part; The antibody of Gpr100 polypeptide; Maybe can encode their nucleic acid.

Preferably, the Gpr100 relevant disease is selected from down group: obesity or weight increase, appetite suppresses, metabolism disorder, diabetes, comprise type i diabetes and type ii diabetes, and associated conditions and body weight associated conditions, being subjected to property of glucose tolerance, insulin resistance syndrome, the X syndrome, peripheral neurophaty, diabetic neuropathy, urinate with the diabetes proteins associated, the lipid metabolism illness, comprise hyperglycemia, hyperlipidemia, dyslipidemia (dyslipidemia), hypertriglyceridemia, acute pancreas adenositis, angiocardiopathy, peripheral artery disease, hypertension, cardiomegaly, the ischemic heart disease, hypercholesterolemia, obesity, and the prevention of obesity or weight increase.

The accompanying drawing summary

Fig. 1 is the figure that shows the result of HMM structure prediction software (http://www.sanger.ac.uk/Software/Pfam/search.shtml) the analyst Gpr100 polypeptide (SEQ IDNO:3) that utilizes pfam.

Fig. 2 is the figure that knocks out plasmid.

Fig. 3 is for showing the figure of the people Gpr100GPCR expression and distribution type that produces by reverse transcription-polymerase chain reaction (RT-PCR).

Fig. 4 shows the Histological section of the white adipose tissue of Gpr100 knock-out mice and wild type contrast.

Fig. 5 shows the figure from the blood sample analysis of Gpr100 animal.

Fig. 5 A is the figure from the blood sample analysis of Gpr100 buck.Fig. 5 B is the figure from the blood sample analysis of Gpr100 jenny.

Fig. 6 be wild type animal and Gpr100 knock-out animal during fasting along with the blood glucose level view of time.

Fig. 7 is the blood sample analysis figure from wild type animal and Gpr100 knock-out animal.

Fig. 8 is the hyperglycemic factor level view of wild type animal and Gpr100 knock-out animal.

Fig. 9 is (16 hours) the wild type animal of demonstration overnight fast and Gpr100 knock-out animal dextrose tolerance test result's figure.

Figure 10 is the figure of overnight fast (16 hours) wild type animal and Gpr100 knock-out animal glucose level.

The figure that Figure 11 analyzes for the horizontal RIA of hyperglycemic factor in the terminal blood sample of (16 hours) wild type that shows overnight fast and Gpr100 knock-out animal.

Figure 12 shows the insulin level of glucose tolerance (GTT) test back in the time of 0,60 and 120 minute.

Insulin level when Figure 13 shows during the fasting 0,6 and 12 hour.

Sequence table

SEQ ID NO:1 shows the cDNA sequence of people Gpr100.SEQ ID NO:2 shows the open read frame that is derived from SEQ ID NO:1.SEQ ID NO:3 shows the amino acid sequence of people Gpr100.SEQ ID NO:4 shows the open read frame of the cDNA of mouse Gpr100.SEQ ID NO:5 shows the amino acid sequence of mouse Gpr100.SEQ ID NO:6-19 shows vector construction body promoter and knockout carrier sequence.

Describe in detail

GPR100GPCR

G protein coupled receptor (GPCR) has been described, especially Orphan G-Protein Coupled Receptors (being called Gpr100 GPCR), its homologue, variant or derivative, with and in treatment, alleviate or diagnosis comprises purposes in the disease of Gpr100 relevant disease such as diabetes and obesity. Of the present invention this will describe in further detail down below with other embodiments.

Gpr100 is also referred to as Gpcr 102 and INSL7 acceptor-2, and shown in the result that the cDNA product to the amplification of encoding human Gpr100 checks order, structurally is associated with other protein of g protein coupled receptor family. The cDNA sequence of SEQ ID NO:1 comprises the open read frame (SEQ ID NO:2, nucleotides numbering 112 to 1039) of 374 amino acid whose polypeptide shown in the coding SEQ ID NO:3. People Gpr100 is found to be positioned human chromosome 1q22.

Unless otherwise noted, enforcement of the present invention will be used chemistry, molecular biology, microbiology, recombinant DNA and the immunologic routine techniques in those of ordinary skills' limit of power. Described technical interpretation is in document. 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 periodic supplements; Current Protocols in Molecular Biology, ch.9,13, and 16, John Wiley ﹠ Sons, New York, N.Y.); B.Roe, J.Crabtree, and A.Kahn, 1996, DNA Isolation and Sequencing:Essential Techniques, John Wiley ﹠ Sons; J.M.Polak and James O ' D.McGee, 1990, In Situ Hybridization:Principles and Practice; Oxford University Press; M.J.Gait (Editor), 1984, Oligonucleotide Synthesis:A Practical Approach, Irl Press; D.M.J.Lilley and J.E.Dahlberg, 1992, Methods of Enzymology:DNA Structure Part A:Synthesis and Physical Analysis of DNA Methods in Enzymology, Academic Press; Using Antibodies:A Laboratory Manual: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, Cold Spring 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); " Immunochemical Protocols, 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 Roskams and 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., and Berkow, R, Eds, ISBN:0911910107, John Wiley ﹠ Sons). This introduces each of these common textbooks by reference at this.

The expression pattern of GPR100

The PCR of Gpr100 cDNA (PCR) the augmentation detection different abundance that Gpr100 expresses in small intestine, lung, kidney, leucocyte and spleen. The expression pattern of Gpr100 GPCR is shown among Fig. 2. Utilize the Gpr100 cDNA of SEQ ID NO:1 by BLASTN seeker EST data source, (BF90022) finds homogeneity in the cDNA in the library that derives from marrow. This shows that Gpr100 normally or in the abnormal structure expresses at these. Therefore, Gpr100 polypeptide, nucleic acid, probe, antibody, expression vector and part can be used for overexpression, not enough detection, diagnosis, treatment and other mensuration of expressing the disease relevant with unconventionality expression in these and other tissues with Gpr100 GPCR.

Of the present invention this will describe in further detail down below with other embodiments.

GPR100 GPCR relevant disease

According to method and composition described here, Gpr100 GPCR can be used for treatment and diagnoses a series of diseases. For simplicity, these diseases are called " Gpr100 relevant disease ".

Therefore, the Gpr100 deficient animals can be used as the model of Gpr100 relevant disease. Gpr100, its fragment, homologue, variant and its derivative, and conditioning agent especially comprise activator and antagonist, can be used for diagnosis or treatment Gpr100 relevant disease. Especially, Gpr100 can be used for screening the molecule that can affect its function, and described molecule can be used for treating the Gpr100 relevant disease.

Here we have confirmed that people Gpr100 is positioned human chromosomal 1q22. Therefore in specific embodiment, Gpr100 GPCR can be used for treatment or diagnosis is positioned this locus, chromosome band, zone, arm or identical chromosomal disease.

Be defined as comprising following (in the bracket for position) with the chromosome position (that is, human chromosomal 1q22) of Gpr100 GPCR is chain in homologous genes seat, chromosome band, zone, arm or chromosomal known disease: epilepsy (1q21), Gaucher disease (Gaucher ' s disease) (1q21), lymphadenomatous progress (lymphoma progression) (1q22), Charcot-Marie-Tooth disease 1B type (1q22), congenital myelin form not enough neuropathy (1q22) and to the neurological susceptibility of family's associating hyperlipidemia (1q22-q23).

Therefore according to preferred embodiment, Gpr100 GPCR can be used for forming not enough neuropathy and to the neurological susceptibility of family's associating hyperlipidemia by any means diagnosis described in the literature or treatment epilepsy, Gaucher disease, lymphadenomatous progress, Charcot-Marie-Tooth disease 1B type, congenital myelin.

As confirming among the embodiment that the knock-out mice of Gpr100 defective presents a series of phenotypes.

In a word, the experiment described in the embodiment has disclosed the impact of Gpr100 acceptor on type ii diabetes and obesity. The Gpr100 deficient mice is subjected to the following process, and comprises the insulin secretion test (GSIST) that GTT, insulin inhibition test (IST) and glucose stimulate. Seen in type ii diabetes, GI may be the result of insulin insensitivity (it absorbs glucose for muscle, fat or liver cell can not respond insulin), or the result of insulin deficit (usually because pancreas beta cell dysfunction), or both. These are measured mouse metabolism and/or store the ability of glucose, blood glucose to the sensitivity of exogenous insulin and the insulin secretion of response glucose. These tests also refer to pay close attention to other observables relevant with diabetes and obesity, such as food intake, metabolic rate, respiratory exchange rate, activity level, body fat composition, Serum Chemical Parameter, for example histology of leptin and relevant organ.

Embodiment shows that Gpr100 mutant animal presents serious hypoglycemia behind the metabolic stress of overnight fast. This became obvious in 6 hours after depriving food.

Therefore, therefore adjusting and Gpr100 deficient animals that Gpr100 participates in glucose can be used as the glucose adjusting, are particularly useful for the model of disease such as the diabetes of glucose insufficiency of accommodation.

Gpr100 can be used for screening the conditioning agent of its function; Described conditioning agent can suffer from the animal of disease such as diabetes. Generally speaking, disclose blood sugar level in the reduction individuality, be preferred for the method for the treatment of diabetes, the method comprises level or the activity that reduces Gpr100 in the individuality. As pointing out that elsewhere this can be by the expression of downward modulation Gpr100 or by using the Gpr100 antagonist to realize.

Embodiment shows that also mutant has the normal tolerance of glucose and do not show the remarkable change of Plasma Glucagon Level. Interrelate with the hypoglycemia phenotype, these results suggest mutant animal dis are in the defective of changing to fatty acid oxidation with the ability that is used for power generation.

Therefore, the Gpr100 deficient animals especially can be used as the model of disease, can't change to be used for power generation as its composition or reason to fatty acid oxidation when hungry in described disease. Gpr100 can be used for screening the molecule that can affect its function, and described molecule can be used for treating described disease.

In addition, after embodiment 3 shows the fasting situation, compare with the control mice of sex coupling with the age, the mutant mouse of isozygotying presents the white adipose tissue and increases and the increase of adipocyte size.

Therefore, the adjusting of Gpr100 participation obesity and Gpr100 deficient animals can be therefore as the obesity models.Gpr100, its fragment, homologue, variant and its derivant, and correctives especially comprise activator and antagonist, can be used for diagnosis or treatment of obesity.Especially, Gpr100 can be used for screening the molecule that can influence its function, and described molecule can be used for treatment of obesity.Generally speaking, disclose body fat in the reduction individuality, be preferred for the method for bariatrician, this method comprises level or the activity that improves Gpr100 in the individuality.As pointing out that elsewhere this can be by the expression of rise Gpr100 or by using the Gpr100 activator to realize.

The Gpr100 relevant disease

Therefore, the Gpr100 relevant disease comprises in following any: obesity, comprise the prevention of obesity or weight increase, and appetite suppresses, metabolism disorder, diabetes.Comprise type i diabetes and type ii diabetes, and the associated conditions illness relevant with body weight, impaired glucose tolerance, insulin resistance syndrome, X syndrome, peripheral neurophaty, diabetic neuropathy, albuminuria, the lipid metabolism illness relevant with diabetes, comprise the prevention of hyperglycemia, hyperlipidemia, dyslipidemia, hypertriglyceridemia, acute pancreatitis, angiocardiopathy, peripheral vascular disease, hypertension, cardiomegaly, ischemic heart disease, hypercholesterolemia, obesity and obesity or weight increase.

As mentioned above, utilize any method and composition described here, Gpr100 GPCR can be used for diagnosing and/or treats any in these specified diseases.In addition, point out that Gpr100 knocks out and has appetite and water uptake and suppress, the compound that therefore can regulate the Gpr100 function can be used as the fat-reducing adjuvant or is used for fat-reducing and fat-reducing plan.

We pay close attention to nucleic acid especially, comprise the carrier of Gpr100 GPCR nucleic acid, polypeptide, pharmaceutical composition, host cell and the transgenic animals that comprise its homologue, variant or derivant, comprise Gpr100 GPCR nucleic acid and/or polypeptide be used for the treatment of or diagnose above the purposes of listed specified disease.In addition, we pay close attention to the compound that can interact or combine with Gpr100 GPCR, the antagonist of preferred Gpr100 GPCR, preferably can reduce the compound of cyclic adenosine monophosphate endogenous levels in the cell, the antibody of Gpr100 GPCR, and preparation or the method for identifying these are in diagnosis or treat purposes in above-mentioned specified disease and the illness or the state of an illness.Especially, we comprise that any of these compound, composition, molecule or the like are used for the treatment of or prevent purposes in the vaccine of specified disease in production.The diagnostic kit that is used for detecting the individuality specified disease is also disclosed.

Evaluation is known in the art by the linkage mapping method of utilizing these or other specified disease that Gpr100 GPCR can treat or diagnosable, and also other local descriptions of the document.

Glucose is regulated

Glucose is necessary for guaranteeing all organ appropriate functional and survival.Though hypoglycemia causes cell death, chronic hyperglycemia also can cause organ or tissue's damage.

Plasma glucose maintains narrow scope, usually 4 and 7mM between, it is produced by liver and is controlled by the balance between peripheral tissues's picked-up and the metabolism from the absorption of intestines by glucose.The response glucose blood plasma level improves, after having meal, and the beta cell excreting insulin of youth's lattice Han Shi pancreas islet.Insulin acts on muscle subsequently and adipose tissue is taken in those cells to stimulate glucose, and act on liver cell to suppress the generation of glucose.

In addition, insulin is stimulating cellular growth and differentiation by stimulating lipogenesis, glycogen and protein synthesis and inhibition lipolysis, decomposition of glycogen and protein decomposition also, and promotes the storage of substrate in fat, liver and muscle.When the blood plasma level of glucose reduces, pancreas A cells secretion hyperglycemic factor, it stimulates glycolysis and glucose in the liver to be released in the blood subsequently.

Diabetes and obesity are disease well known in the art.Every kind summary description is as follows:

Diabetes

Diabetes are defined as the state that wherein carbohydrate and lipid metabolism are regulated irrelevantly by insulin.Two kinds of principal modes of diabetes have been identified, I and II type.Type i diabetes is represented the not too general form of this disease, involves the diabetic of 5-10%.Think that its autoimmunity that is produced the beta cell of insulin by youth's lattice Han Shi pancreas islet destroys institute and causes.The external source administration of insulin alleviates this pathologic, physiologic usually.Type ii diabetes is by the modal form of this disease and may be caused by the associating defective of the insulin secretion and the mechanism of action.Two kinds of forms, I type and II type have similar complication, but different Pathological Physiology.

First phase characteristic of type ii diabetes is that muscle and/or other organs can't be replied insulin normal circulation concentration.This is relevant with obesity, sedentary lifestyle and/or genetic predisposition usually.Be the raising of pancreas beta cell insulin secretion subsequently, this state of an illness is called hyperinsulinemia.Finally, the pancreas beta cell no longer can compensate, and causes impaired glucose tolerance, chronic hyperglycemia and tissue damage.The sophisticated signal approach that participates in blood glucose adjusting and metabolism provides some potential targets, is used for the treatment of the abnormal glucose metabolism state of an illness such as type ii diabetes or obesity.

Obesity

Obesity is influence at least three thousand nine hundred ten thousand American diseases: promptly surpassing among five children of 1/4th peace treaties among all adults has one.In every year, obesity causes at least that in the U.S. extra death of at least 300,000 example and cost country are above 100,000,000,000 dollars.In past 10 years, the obesity population ratio of the U.S. is increased to 32% from 25%.Obesity is measured by body mass index or BMI, and it is to be used for determining a people whether obesity or overweight mathematical computations.BMI is by with body weight for humans kilogram number square calculating divided by its height rice number.30 or bigger BMI think and be obesity that and the BMI of 25-29.9 thinks for overweight.Yet diagnostic criteria may cause misleading for have more polymyarian cube meat and the still less people of body fat than the ordinary people as the sportsman.Surpass 70,000,000 Americans and be considered to overweight.

Health problem includes but not limited to the cancer and the osteoarthritis of angiocardiopathy, blood pressure, type ii diabetes, high cholesterol, gout, some type, and is relevant with the overweight state of an illness and obesity.

The homogeneity of GPR100 and similarity

G protein coupled receptor SALPR somatostatin and angiotensins sample peptide acceptor.(homogeneity=141/322 (43%), positive=194/322 (59%)).

Use the HMM structure prediction software (http://www.sanger.ac.uk/Software/Pfam/search.shtml) of pfam to analyze the GPCR (referring to Fig. 1) of Gpr100 polypeptide (SEQ IDNO:3) confirmation Gpr100 peptide as the 7TM-1 structural group.

Cloned people the mouse homologue of Gpr100 GPCR, and its nucleotide sequence and amino acid sequence are shown as SEQ ID NO:4 and SEQ ID NO:5 respectively.The mouse Gpr100GPCR cDNA of SEQ ID NO:4 shows the height homogeneity (homogeneity=571/693 (82%)) with people Gpr100 GPCR (SEQ ID NO:2) sequence, and the amino acid sequence of mouse Gpr100 GPCR (SEQ ID NO:5) shows and the height homogeneity of people Gpr100 GPCR (SEQ ID NO:3) and similarity (homogeneity=235/379 (62%), positive=264/379 (69%)).

Therefore people and mouse Gpr100 GPCR are the member of g protein coupled receptor (GPCR) extended familys.

GPR100 GPCR polypeptide

As used herein, term " Gpr100 GPCR polypeptide " is used to refer to the polypeptide that comprises amino acid sequence shown in SEQ ID No.3 or the SEQ ID NO:5, or its homologue, variant or derivant.Preferably, polypeptide comprises homologue, variant or the derivant of sequence shown in the SEQ ID NO:3.

" polypeptide " refers to comprise two or more amino acid whose any peptide or the protein that the peptide bond by peptide bond or modification mutually combines, i.e. peptide isostere." polypeptide " both referred to short chain, and so-called peptide, oligopeptides or oligomer also refer to long-chain, so-called protein.Polypeptide can comprise the amino acid amino acid in addition of 20 encoding genes.

" polypeptide " comprises the amino acid sequence of for example translating back processing modification by natural process, or the amino acid sequence of modifying by chemical modification technology known in the art, these modification techniques are described in the basic textbook preferably, be described in greater detail in the monograph, in the research document of treatise, description arranged also.Modification can betide any zone of polypeptide, comprises peptide main chain, amino acid side chain and amino or carboxyl terminal.Be to be understood that in given polypeptide the modification of same type can exist with different degree at same loci or in several sites.And given polypeptide can comprise polytype modification.

As the result of ubiquitination, polypeptide can be a branch; Can be ring-type also, have or do not have branch.Ring-type, branch and branch's ring type polypeptide can produce from translation back natural process, maybe can prepare by synthetic method.Modification comprises acetylation; acidylate; the ADP-ribosylation; amidation; the covalent bond of flavine; the covalent bond of heme part; the covalent bond of nucleotide or nucleotide derivative; the covalent bond of lipid or lipid derivant; the covalent bond of phosphatidylinositols; crosslinked; cyclisation; form disulfide bond; demethylation; form covalent cross-linking; form cystine; form pyroglutamic acid; formylation; the gamma-carboxylation effect; glycosylation; the GPI anchor forms; hydroxylation; iodination; methylate; myristoylation; oxidation; proteolysis processing; phosphorylation; isoprenylation; racemization; selenoylation; sulfation; transfer-RNA mediation in protein, increase amino acid, for example arginylization and ubiquitination.Referring to, for example, Proteins-Structureand Molecular Properties, 2nd Ed., T.E.Creighton, W.H.Freeman andCompany, New York, 1993 and Wold, F., Posttranslational ProteinModifications: Perspectives and Prospects, pgs.1-12 in PosttranslationalCovalent Modification of Proteins, B.C.Johnson, Ed., Academic Press, NewYork, 1983; Seifter et al., " Analysis for protein modifications and nonproteincofactors ", Meth Enzymol (1990) 182:626-646 and Rattan et aL, " ProteinSynthesis:Posttranslational Modifications and Aging ", Ann NY AcadSci (1992) 663:48-62.

The term that this paper relates to " variant ", " homologue ", " derivant " or " fragment " comprise sequence are carried out any replacement, change, modification, replacement, disappearance or added (or a plurality of) amino acid.Unless other implication of approval mentions that " Gpr100 " and " Gpr100GPCR " comprises this variant, homologue, derivant and the fragment of mentioning Gpr100 in the context.

Preferably, for Gpr100, the amino acid sequence that obtains has the GPCR activity, more preferably at least with have the identical activity of Gpr100 GPCR shown in SEQ ID NO:3 or the SEQ ID NO:5.Especially, the homogeneity on structure and/or the function contained in term " homologue ", as long as the amino acid sequence that obtains has the GPCR activity.About 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.More preferably have at least 95%, more preferably at least 98% sequence homogeneity.These terms also comprise the amino acid whose polypeptide that is derived from Gpr100 GPCR nucleotide sequence allelic variant.

When " receptor active " of mentioning acceptor such as Gpr100 GPCR or " biologic activity ", these terms are used to refer to the metabolism or the physiological function of Gpr100 acceptor, comprise the active of similar active or improvement or have these activity of the adverse side effect of reduction.The antigenicity and the immunogenic activity that also comprise the Gpr100 acceptor.The example of GPCR activity and detection and quantitative these active methods are known in the art, and describe in detail in other parts of the present invention.

" disappearance " is defined as the change of nucleotide and amino acid sequence as used herein, wherein is respectively one or more nucleotide and amino acid residue disappearance.As used herein " insertion " or " interpolation " refer to the change of nucleotide or amino acid sequence, it causes comparing with naturally occurring material, is respectively the interpolation of one or more nucleotide or amino acid residue." replacement " is that one or more nucleotide or amino acid are respectively by the result of different nucleotide or aminoacid replacement as used herein.

The Gpr100 polypeptide also can have disappearance, insertion or the replacement of amino acid residue, and it produces reticent change and causes the suitable amino acid sequence of function.Planned aminoacid replacement can based on the polarity of residue, electric charge, dissolubility, hydrophobicity, water wettability and/similarity of amphiphilic character etc. carries out.For example, electronegative amino acid comprises aspartic acid and glutamic acid; The amino acid of positively charged comprises lysine and arginine; The amino acid that have similar hydrophilicity value, has a uncharged polar head group comprises leucine, isoleucine, valine, glycocoll, alanine, asparagine, glutamine, serine, threonine, phenylalanine and tyrosine.

For example can guard replacement according to following table.Amino acid in the second hurdle same unit, preferably can replace mutually with the amino acid in the delegation in third column.

Aliphatic	Nonpolar	GAP
					ILV
	Polarity-uncharged	CSTM
					NQ
	Polarity-charged	DE
					KR
Aromatic		HFWY

The Gpr100 polypeptide can further comprise the allogeneic amino acid sequence, usually at N-end or C-end, and preferred N-end.Heterologous sequence can comprise to be influenced in the cell or the sequence (for example targeting sequencing) of extracellular protein target.Heterologous sequence also can comprise the sequence that improves the Gpr100 immunogenicity of polypeptides and/or be convenient to polypeptide evaluation, extraction and/or purifying.Another particularly preferred heterologous sequence is the amino acids sequence, for example is preferably the polyhistidine of N-end.At least 10 amino acid, preferably at least 17 amino acid but to be less than 50 amino acid whose polyhistidine sequences be particularly preferred.

Gpr100 GPCR polypeptide can be the form of " maturation " protein, perhaps can be than the larger protein part of fusion for example.Comprise that the additional amino acid sequence usually is favourable, described additional amino acid sequence contains the sequence such as the polyhistidine residue of secretion or targeting sequencing, presequence, aided purification, or is used for stable appended sequence in the recombinant production process.

By recombination method, use known technology to prepare the Gpr100 polypeptide easily.Yet, also can pass through synthetic method, use for example solid phase synthesis preparation of the known technology of person skilled in the art.The Gpr100 polypeptide also can be made into fusion, for example with assisted extraction and purifying.The example of fusion gametophyte comprises glutathione-S-transferase (GST), 6xHis, GAL4 (DNA combination and/or transcription activating functional domain) and beta galactosidase.Comprise the proteolysis restriction enzyme site between fusion gametophyte and destination protein sequence, for example the fibrin ferment restriction enzyme site also is easily, to allow to remove the fusion sequence.Preferred fusion can not hinder the protein function of aim sequence.

The Gpr100 polypeptide can be the form of separating basically.This term is meant by manually transforming from native state.If " separation " composition or material exist in nature, it is changed or removes from its initial environment, and perhaps the two has concurrently.For example, natural polynucleotide, nucleic acid or the polypeptide that is present in the animal body alive is not " separation ", but the identical polynucleotide, nucleic acid or the polypeptide that separate from the coexistence raw material of its native state are " separation ", as the term that adopts herein.

Yet, be to be understood that the Gpr100 gpcr protein can with carrier or mixing diluents that can not the interferencing protein Expected Results, and still think to separate basically.The Gpr100 polypeptide can also be the form of purifying basically, and in this case, it comprises the protein that is present in the goods usually, surpasses 90% in the wherein said goods, and for example 95%, 98% or 99% protein is Gpr100 GPCR polypeptide.

Presents also relates to the peptide of the part that comprises the Gpr100 polypeptide.Therefore, the fragment and homologue, variant or the derivant that comprise Gpr100 GPCR.Described peptide length can be between 2 and 200 amino acid, preferably between 4 and 40 amino acid.Described peptide can be derived from Gpr100 GPCR polypeptide disclosed herein, for example by with suitable enzyme for example trypsase digest.Perhaps peptide, fragment etc. can be synthetic by recombinant methods or synthetic method.

Term " peptide " comprises the variant of various synthetic peptides known in the art, for example retroinverso D peptide.This peptide can be antigenic determinant and/or T-cell epitope.This peptide can have immunogenicity in vivo.Preferred this Toplink is induced neutralizing antibody in vivo.

From the GPCR sequence of different plant species, can determine which zone of amino acid sequence is (" the homology zone ") of guarding between the variety classes by contrast, and between the variety classes which zone change (" allos zone ").

Therefore, the Gpr100 polypeptide can comprise the sequence corresponding at least a portion homology zone.The homology zone shows the homology of height between at least two species.For example, use the above-mentioned method of inspection, the homology zone can show at least 70% on amino acid levels, preferably at least 80%, more preferably at least 90%, even more preferably at least 95% homogeneity.As hereinafter more elaborating, the peptide that comprises corresponding to the sequence in homology zone can be used for therapeutic strategy.Perhaps, Gpr100 GPCR peptide can comprise the sequence corresponding at least a portion allos zone.The allos zone shows the low homology degree between at least two species.

GPR100 GPCR polynucleotide and nucleic acid

As describing in further detail, the present invention includes Gpr100 polynucleotide, Gpr100 nucleotide and Gpr100 nucleic acid, production method and these purposes etc. in this paper other places.

Term " Gpr100 polynucleotide ", " Gpr100 nucleotide " and " Gpr100 nucleic acid " are used interchangeably, and be used to refer to the polynucleotide/nucleic acid that comprises the nucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:2 or the SEQ ID NO:4, or its homologue, variant or derivant.Preferably, polynucleotide/nucleic acid comprises or is nucleic acid sequence SEQ ID NO:1 or SEQ ID NO:2, the most preferably homologue of SEQ ID NO:2, variant or derivant.

These terms also comprise the nucleotide sequence of can encode Gpr100 polypeptide and/or peptide.Therefore, Gpr100GPCR polynucleotide and nucleic acid comprise encoding and comprise the nucleotide sequence of the polypeptide of amino acid sequence shown in SEQ ID NO:3 or the SEQ ID NO:5, or its homologue, variant or derivant.Preferably, Gpr100 GPCR polynucleotide and nucleic acid comprise encoding and comprise the nucleotide sequence of the polypeptide of amino acid sequence shown in the SEQ ID NO:3, or its homologue, variant or derivant.

" polynucleotide " are often referred to any polyribonucleotide or polydeoxyribonucleotide, and it can be the RNA of unmodified or the RNA or the DNA of DNA or modification." polynucleotide " include, without being limited to list and double-stranded DNA, be the DNA of the potpourri of strand and double-stranded region, strand and double-stranded RNA, be the RNA of the potpourri of strand and double-stranded region, the crossbred that comprises DNA and RNA, described DNA and RNA can be strands, or more typically, are potpourris double-stranded or strand and double-stranded region.In addition, " polynucleotide " refer to comprise the three chain zones that RNA or DNA or RNA and DNA comprise.The term polynucleotide also comprise DNA and the RNA that contains one or more modified bases, and because of stable or adorned DNA of other reason main chain or RNA." modification " base comprises, for example tritylation base and rare bases such as trophicardyl.DNA and RNA have carried out multiple modification; Therefore, " polynucleotide " comprise the polynucleotide of chemistry, enzymatic or the metabolism modified forms found usually under natural situation, and the chemical species of virus and peculiar DNA of cell and RNA." polynucleotide " also comprise short relatively polynucleotide, are often referred to as oligonucleotides.

The technician should be appreciated that the result of genetic codon degeneracy, makes the identical polypeptide of many nucleotide sequence codifieds.

As used herein, term " nucleotide sequence " refers to nucleotide sequence, oligonucleotide sequence, polynucleotide sequence and variant thereof, homologue, fragment and derivant (as its part).Nucleotide sequence can be the DNA or the RNA of genomic or synthetic or recombinant sources, and it can be double-stranded or strand, and no matter whether represent sense strand or antisense strand or its combination.The term nucleotide sequence can use recombinant DNA technology preparation (for example, recombinant DNA).

Preferably, term " nucleotide sequence " refers to DNA.

The term that this paper relates to " variant ", " homologue ", " derivant " or " fragment " comprise from or carry out any replacement, change, modification, substitute, lack or add (or a plurality of) nucleic acid to the sequence of Gpr100 nucleotide sequence.Unless context is approved in addition, is mentioned that " Gpr100 " and " Gpr100GPCR " comprises described variant, homologue, derivant and the fragment of mentioning Gpr100.

Preferably, acquisition nucleotide sequence coded has GPCR activity, the polypeptide that preferably has the activity identical at least with the GPCR shown in SEQ ID NO:3 or the SEQ ID NO:5.Preferably, term " homologue " intention contains the homogeneity of structure and/or function, the nucleotide sequence coded polypeptide with GPCR activity that makes acquisition.Homogeneity (being similarity) about sequence preferably has at least 70%, more preferably at least 75%, more preferably at least 85%, more preferably at least 90% sequence homogeneity.More preferably have at least 95%, more preferably at least 98% sequence homogeneity.These terms also comprise the allelic variant of this sequence.

The calculating of sequence homology

About the sequence homogeneity of any sequence of occurring here can by with simple " eyeball (eyeball) " of any one or a plurality of sequence and another sequence relatively (that is, strictness is compared) other sequence and one or more sequence whether have for example at least 70% sequence homogeneity.

Relatively sequence homogeneity also can be determined by commercially available computer program, and described computer program can use default parameter for example, be used for any appropriate algorithm of definite homogeneity calculates % homogeneity between two or more sequences.The representative instance of this computer program is CLUSTAL.Be used for determining that homogeneity between two sequences and other computer program means of similarity include but not limited to GCG routine package (Devereux et al 1984 Nucleic Acids Research 12:387) and FASTA (Atschul et al 1990 J Molec Biol 403-410).

The % homology can be calculated adjoining on the sequence,, a sequence and another sequence is carried out sequence alignment that is, and each amino acid in sequence directly carries out with the corresponding amino acid of another sequence, relatively whenever next residue.This is called as " unnotched " sequence alignment.Usually, described unnotched contrast is only carried out on the residue of relatively small amount.

Although this is very simple and consistent method, but it is not considered, for example, the identical sequence centering of others, one is inserted or disappearance will cause amino acid residue subsequently to be evicted from sequence alignment, therefore when carrying out the integral body comparison, may cause the decline significantly of % homology.Therefore, most sequence comparative approach are designed to produce best comparison, and described best comparison is considered possible insertion and disappearance and can be to the whole homology point penalty inadequately of marking.This attempts to maximize local homology by insertion " breach " in the sequence contrast and realizes.

But, these more complicated methods have been specified " breach point penalty " to each breach that occurs in the sequence contrast, like this, for the same amino acid of equal number, the sequence contrast (two more high correlations that are compared between the sequence are answered in its reflection) that has the least possible breach will obtain higher scoring than the sequence that many breach are arranged.General use " outbreeding breach cost (Affine gap costs) " comes the high relatively cost of existence button (charge) to breach, gives each less point penalty of residue button subsequently in the breach.This is the breach points-scoring system that the most generally uses.High breach point penalty has generation the optimized sequence contrast of less breach certainly.Most sequence contrast programs allow to revise the breach point penalty.But when using this software to do the sequence comparison, preferably use default value.For example, when using GCG Wisconsin Bestfit routine package, the default breach point penalty of amino acid sequence is that breach is-12, each extends to-4.

Therefore the calculating of maximum % homology at first needs to produce the best comparison of considering the breach point penalty.The suitable computer program that carries out this comparison is GCG Wisconsin Bestfit routine package (Universityof Wisconsin, U.S.A.; Devereux et al., 1984, Nucleic Acids Research 12:387).The example that can carry out other softwares of sequence comparison includes but not limited to, blast program bag (Ausubel etal., 1999 ibid-Chapter 18), FASTA (Atschul et al., 1990, J.Mol.Biol., 403-410) and the compare tool of GENEWORKS series (suite).BLAST and FASTA are available, to be used for off-line and on-line search (Ausubel et al., 1999 ibid, pages 7-58 to 7-60.

Although final % homology can enough homogeneity be measured, comparison process itself is not the paired comparisons based on all or none usually.On the contrary, use the similar rating matrix of classification usually, being that each relatively specifies scoring in pairs according to chemical similarity or evolutionary distance.The example of normally used this matrix is the BLOSUM62 matrix, and it is the default matrix of BLAST series of programs.GCG Wisconsin program is used public default value usually, if perhaps provide, can use self-defined symbol comparison sheet.The preferred public default value that uses in the GCG routine package, or under the situation of using other softwares, use default matrix, for example BLOSUM62.

Advantageously, use BLAST algorithm parameter to be set to default value.Describe the BLAST algorithm in detail in http://www.ncbi.nih.gov/BLAST/blast help.html, it is hereby incorporated by.Search parameter also can be advantageously provided and be defined default parameter.

Advantageously, when assessing by BLAST, " same basically " is equal to the sequence of being mated with about at least 7 EXPEXT value, preferably be at least about 9, and most preferably 10 or more than.In the blast search, the default threshold value of EXPECT normally 10.

BLAST (Basic Local Alignment Search Tool) is the heuristic search algorithm that program blastp, blastn, blastx, tblastn and tblastx use; These programs are used statistical method (Karlin and Altschul 1990, the Proc.Natl.Acad.Sci.USA 87:2264-68 of 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 carry out some and improve to give their Search Results conspicuousness.Blast program is transformed to be applicable to the sequence similarity search, for example identifies the homologue of search sequence.To the discussion of basic problem in the similarity searching of sequence library referring to Altschul et al (1994) Nature Genetics 6:119-129.

Can from // five blast programs that www.ncbi.nlm.nih.gov obtains carry out following task: blastp-with amino acid search sequence and protein sequence database comparison; Blastn-compares nucleotide query sequence and nucleotide sequence database; Blastx-compares six frames (six-frame) the concept nature translation product and the protein sequence database of nucleotide query sequence (two chain); Tblastn-compares protein search sequence and nucleotide sequence database, and described nucleotide sequence database is read frames with whole six and dynamically translated (two chain); Tblastx-compares the six frames translation thing of nucleotide query sequence and the six frames translation thing of nucleotide sequence database.

BLAST utilizes following search parameter:

HISTOGRAM-shows the scoring histogram of each search; Default to yes.(referring to the Parameter H of BLAST guide).

DESCRIPTIONS-limits the quantity to the Short Description of the matching sequence that reports to specified numeral; Defaultly be restricted to 100 descriptions.(referring to the V parameter in the man pages).

The EXPECT-report is at the significance,statistical threshold value of the coupling of database sequence; Default value is 10, therefore makes according to Karlin and Altschul (1990) stochastic model, and 10 couplings are expected only be found accidentally.If the significance,statistical of giving coupling is greater than the EXPECT threshold value, this coupling will not be in the news.Lower EXPECT threshold value is strict more, causes proportioning at random still less to be reported.Fractional value is acceptable (referring to the parameter E in the BLAST handbook).

CUTOFF---report high score fragment right block score.Calculate default value (seeing above) from the EXPECT value.Only when the significance,statistical of the HSP that gives database sequence at least with to give independently HSP that score equals the CUTOFF value the same when high, the HSP of report database sequence.Higher CUTOFF value is stricter, causes less coupling at random by report (referring to the parameter S in the BLAST handbook).Typically, the conspicuousness threshold value can use EXPECT to control more intuitively.

ALIGNMENTS---database sequence is restricted to reporting that high score fragment is to (HSPs) specified quantity; Default restriction is 50.If satisfy the significance,statistical threshold value of reporting (referring to following EXPECT and CUTOFF) by chance than this more database sequence, only report is endowed the coupling (referring to the B parameter in the BLAST handbook) of maximum significance,statistical.

MATRIX---for BLASTP, BLASTX, TBLASTN and TBLASTX specify interchangeable rating matrix.Default matrix is BLOSUM62 (Henikoff ﹠amp; Henikoff, 1992).Effective interchangeable selection comprises: PAM40, PAM120, PAM250 and IDENTITY.There is not interchangeable rating matrix to can be used in BLASTN; Specifying the MATRIX instruction to obtain makeing mistakes in the BLASTN request replys.

STRAND---the TBLASTN search is limited to the only top or the bottom chain of database sequence; Perhaps BLASTN, BLASTX or TBLASTX search are limited to the only top of search sequence or the frame on the chain of bottom.

FILTER---shelter as using Wootton ﹠amp; The fragment of the search sequence with low combinatorial complexity that the SEG program of Federhen (1993) Computers andChemistry 17:149-163 is measured is perhaps as using Claverie ﹠amp; The fragment of forming by short period property internal repeat of the XNU program determination of States (1993) Computers and Chemistry 17:191-201, perhaps for BLASTN use Tatusov and Lipman the DUST program (referring to Http: //www.ncbi.nlm.nih.gov).Filtration can be removed from blast output on the statistics significantly but biologically insignificant report (for example at common acidity-, alkalescence-or the Query Result in the abundant zone of proline), stay and can be used for carrying out zone specific coupling, that have more biological significance in the search sequence at database sequence.

In nucleotide sequence, use letter " N " to replace (for example, " NNNNNNNNNNNNN ") by the low-complexity sequence that filter is found, and in protein sequence, use letter " X " to replace low-complexity sequence (for example " XXXXXXXXX ").

Filtration only is used for search sequence (or its translation product), is not suitable for database sequence.Default filtration is DUST for BLASTN, is SEG for other program.

When being used for the sequence of SWISS-PROT, it is normal being sheltered by SEG, XNU or the two without any thing, so expectation is filtered and can be told on.In addition, in some cases, sequence is masked fully, shows that the significance,statistical of any coupling of reporting at unfiltered search sequence is shady.

NCBI-gi-causes that except that going into to hide registration and/or the locus title NCBI gi identifier is shown in output.

Most preferably, sequence is relatively used Http:// www.ncbi.nlm.nih.gov/BLASTOn the simple blast search algorithm that provides carry out.In some embodiments, when measuring sequence homogeneity, do not use the breach point penalty.

Hybridization

This paper also comprise can with the nucleotide sequence of the sequence hybridization that proposes here, perhaps its any fragment or derivant are perhaps with the nucleotide sequence of the complement hybridization of any above-mentioned substance.

Hybridization is meant " process (Coombs J (1994) Dictionary of Biotechnology; Stockton Press; New York NY) that nucleic acid chains and complement chain combine by base pairing and as the amplification procedure that carries out with polymerase chain reaction technique of description in Dieffenbach CW and GS Dveksler (1995; PCR Primer; a Laboratory Manual; Cold Spring Harbor Press, Plainview NY).

As Berger and Kimmel (1987, Guide to Molecular Cloning Techniques, Methods in Enzymology, 152 volumes, Academic Press, San Diego CA) lectured in, hybridization conditions depends on the melting temperature (Tm) of nucleic acid in conjunction with compound, and gives definite " severity " as hereinafter illustrating.

The nucleotide sequence that can propose with this paper or the nucleotide sequence of their complementary strand selective cross, corresponding nucleotides sequence common and that this paper proposes is listed at least 20, preferably at least 25 or 30, for example at least 40,60 100 or the zone of more continuous nucleotides have at least 70%, preferably at least 75%, more preferably at least 85% or 90%, even at least 95% or 98% homology more preferably.Preferred nucleotide sequence will comprise the NO:1 with SEQ ID, and the zone of 2 or 4 homologies preferably has at least 70%, 80% or 90% and more preferably at least 95% homology with one of them sequence.

Term " selective cross " is meant that the nucleotides sequence as probe is listed in and finds to be used under the condition that target nucleotide sequences and probe hybridize with the level that is significantly higher than background.Background hybridization can take place because of the existence of other nucleotide sequence, for example in screened cDNA or genome dna library.In this incident, background is meant by the signal level that interact to produce between the non-specific DNA member in probe and library, this level with compare low 10 times with the interactional intensity of the observed specificity of target DNA, preferably hang down 100 times.Can measure interactional intensity, for example, for example use by the radiolabeled probe ³²P.

The nucleotide sequence of the nucleotide sequence hybridization that can propose with this paper to the stringent condition in moderate is also included within this paper scope.As Berger and Kimmel (1987, Guide to MolecularCloning Techniques, Methods in Enzymology, 152 volumes, Academic Press, SanDiego CA) lectured in, hybridization conditions depends on the melting temperature (Tm) of nucleic acid in conjunction with compound, and gives definite " severity " as hereinafter illustrating.

Stringent condition typically occurs in Tm-5 ℃ (being lower than 5 ℃ of probe Tm); High stringent condition occurs in and is lower than about 5 ℃-10 ℃ of Tm; The moderate stringent condition occurs in and is lower than about 10 ℃-20 ℃ of Tm; Low stringency condition occurs in and is lower than about 20 ℃-25 ℃ of Tm.The person skilled in the art should be appreciated that the strictest hybridization can be used for differentiating or detecting identical nucleotide sequence, and the strict hybridization of moderate (or low) can be used for differentiating or detecting similar or relevant nucleotide sequence.

In preferred embodiments, we have described can be under stringent condition (for example 65 ℃ and 0.1xSSC{1xSSC=0.15M NaCl, 0.015M sodium citrate pH 7.0}) and the nucleotide sequence of one or more Gpr100 GPCR nucleotide sequence hybridizations.When nucleotide sequence is two strands, double-helical two chains, no matter it is separately or combination, all is included in this paper.When nucleotide sequence is strand, be to be understood that the complementary series of this nucleotide sequence is also included within the scope of this paper.

The disclosure also comprise can with the nucleotide sequence of the sequence hybridization of the sequence complementation that herein occurs, or its any fragment or derivant.Same, the disclosure comprise with can with the nucleotide sequence of the sequence complementation of correlated series hybridization.These nucleotide sequence types are examples of variant nucleotide sequence.Aspect this, term " variant " comprise with can with the sequence of the sequence complementation of the nucleotide sequence hybridization that herein occurs.Yet, term " variant " preferably include with can be under stringent condition (for example 65 ℃ and 0.1 * SSC{1 * SSC=0.15M NaCl, 0.015 trisodium citrate pH 7.0}), with the sequence of the nucleotide sequence hybridization that the occurs sequence of complementation mutually herein.

The clone of GPR100 GPCR and homologue

The disclosure also comprises the nucleotide sequence of the sequence that is complementary to this paper proposition, or its any fragment or derivant.Be complementary to its fragment as infructescence, this sequence can be used as and differentiates and the probe of cloning similar GPCR sequence in other organism so.

Therefore this paper makes that for example relevant gene is achieved on clone Gpr100GPCR, its homologue and other structures or the functions such as mouse, pig, sheep from people and other species.With the same or enough same polynucleotide of nucleotide sequence that comprise in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4 or its fragment, the hybridization probe that can be used as cDNA and genomic DNA comes from part or full-length cDNA and the genomic clone of the library separation coding Gpr100 GPCR that is fit to.Described probe also can be used for separating and the Gpr100 gpcr gene has sequence similarity, preferably has the cDNA and the genomic clone (comprising homologue or the straight gene to homologue of coding from the species except that human) of other genes of height sequence similarity.Screening by hybridization, clone and sequencing technologies are well known by persons skilled in the art, and describe in (the same) such as for example Sambrook.

It is 70% same, preferred 80% same, more preferably 90% same even more preferably 95% same generally to be suitable as the nucleotide sequence of probe and object of reference.This probe generally comprises at least 15 nucleotide.Preferred described probe has at least 30 nucleotide, and can have at least 50 nucleotide.Particularly preferred probe between 150 to 500 nucleotide scopes, more particularly about 300 nucleotide.

In one embodiment, polynucleotide for the Gpr100 GPCR polypeptide that obtains to encode, comprise from the homologue of the species except that human with directly to homologue, comprise with having SEQ ID NO:1, SEQ ID NO:2, the label probe of SEQ ID NO:4 or its fragment screens suitable library under stringent hybridization condition, and separation contains the part of described polynucleotide sequence or the step of full-length cDNA and genomic clone.Described hybridization technique is well known by persons skilled in the art.Stringent hybridization condition is as top definition, or it is replaceable at 42 ℃, in comprising following solution, be incubated overnight: 50% formamide, 5 * SSC (150mM NaCl, 15mM trisodium citrate), 50mM sodium phosphate (pH 7.6), 5 * Denhardt ' s solution, the salmon sperm DNA of 10% dextran sulfate and 20 micrograms/ml sex change, shearing, afterwards in 0.1 * SSC in about 65 ℃ of washing nozzles.

The functional selection of GPR100 GPCR

Clone's the Gpr100 GPCR polynucleotide of inferring can be by sequential analysis or functional selection checking.For example, Gpr100 GPCR that infers or homologue can following analysis receptor actives.The establishing criteria program with the external linearization plasmid template of synthesizing to come own coding Gpr100 receptor cdna of RNA polymerase add the cap rna transcription this.Cell-free transcription folder is that 0.2mg/ml is suspended in the aqueous solution with the final concentration.Remove the removal ovary leaf from the female toad that grows up, what obtain stage V removes the vesica egg mother cell, uses microinjector with 50nl particle (bolus) injection rna transcription this (10ng/ egg mother cell).Use the voltage clamp of two electrodes to measure the electric current that individual Africa xenopus egg mother cell response contacts activator.In the Barth ' of no Ca2+ s nutrient culture media, at room temperature carry out record.As described in further detail below, the Africa xenopus system also can be used for screening the tissue/cell extract of known part and activation part.

The expression analysis of GPR100 GPCR

In order to design effective therapy of treatment Gpr100 GPCR relevant disease, determine that the expression pattern of Gpr100 (no matter wild type or concrete mutant) is useful.Therefore, methods known in the art can be used to determine to express organ, tissue and the cell type (and stage of development) of Gpr100.For example, can carry out Northern conventional or " electronics ".Also can use reverse transcriptase PCR (RT-PCR) to detect the expression of Gpr100 gene or mutant.Be used for determining that the more sensitive method of Gpr100 expression pattern comprises that the RNAse protection is analyzed as is known to persons skilled in the art.

It is to be used to detect the laboratory technique that gene transcripts exists that Northern analyzes, and relates to the nucleotide sequence of mark and the film that has combined on it from the RNA of particular cell types or tissue is hybridized.(Sambrook，supra，ch.7 and Ausubel，F.M.，et al.supra，ch.4 and 16.)。The similar computer technology (" electronics Northern ") of using BLAST is used in such as the same or relevant molecule of search in the nucleosides database of GenBank or LIFESEQ database (Incyte Pharmaceuticals).The advantage of this type analysis is that it may be more quicker than the hybridization based on multiple film.And the sensitivity that can improve computer search determines whether any concrete coupling is classified as accurate or homology.

Describe in further detail as this paper other places, comprise that the polynucleotide of above-mentioned probe and polypeptide can be used as research reagent and the material of finding treatment and diagnosis animal and human disease.

GPR100 GPCR polypeptide expression

The disclosure comprises the method that produces Gpr100 GPCR polypeptide.This method generally comprises under suitable condition (that is, expressing the condition of Gpr100 GPCR polypeptide) and cultivates the nucleic acid that comprises coding Gpr100 GPCR polypeptide, or the host cell of its homologue, variant or derivant.

In order to have expressed bioactive Gpr100 GPCR, nucleotide sequence or its homologue, variant or the derivant of coding Gpr100 GPCR are inserted into suitable expression, promptly comprise the essential element of carrier transcribe and translate to(for) the coded sequence that inserts.

The method of well known to a person skilled in the art is used for making up the sequence that contains coding Gpr100 GPCR and suitable expression vector of transcribing with the translational control element.These methods comprise extracorporeal recombinant DNA technology, synthetic technology and vivo gene reorganization.These technology are described among the J. etc. at Sambrook.(1989；Molecular Cloning，A Laboratory Manual，ch.4，8，and 16-17，Cold SpringHarbor Press，Plainview，N.Y.)and Ausubel，F.M.et al.(1995 and periodicsupplements；Current Protocols in Molecular Biology，ch.9，13，and 16，JohnWiley & Sons，New York，N.Y.).

Multiple expression vector/host system can be used to comprise and express the sequence of coding Gpr100 GPCR.These include but not limited to, microorganism, for example 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.The present invention is not subjected to the restriction of used host cell.

" controlling element " or " regulating and controlling sequence " is the carrier non-translational region transcribing and translate (be enhancer, promoter and 5 ' and 3 ' non-translational region) that interacts of the cell protein with the host.The length of these elements can be different with specificity.Carrier system and the host of depending on use can utilize any amount of suitable element of transcribing and translate, and comprise the promoter of composing type and induction type.For example when in bacterial system, cloning, can utilize the promoter of induction type, for example the BLUESCRIPT phasmid (Stratagene, La Jolla, Calif.) or the heterozygosis LacZ promoter of PSPORT1 plasmid (GIBCO/BRL) etc.Baculovirus polyhedrin body protein promoter can be used in the insect cell.The promoter of plant-derived cellular genome (for example heat shock, RUBISCO and storage protein gene) or plant-derived virus or enhancer (for example viral promotors or targeting sequencing) can be cloned in the carrier.In mammal cell line system, be preferred from the promoter of mammalian genes or mammalian virus.If must produce the clone of a plurality of copies that contain coding Gpr100 GPCR sequence, can use carrier based on SV40 or EBV with suitable selected marker.

In bacterial system, can select many expression vectors according to the desired use of Gpr100 GPCR.For example, when a large amount of Gpr100 GPCR of needs induces antibody, but the carrier of the high level expression of the fusion that instruction easily is purified.Described carrier includes but not limited to, multi-functional escherichia coli cloning and expression vector, as BLUESCRIPT (Stratagene), the sequence of Gpr100GPCR of wherein encoding can be connected in the carrier, be in the reading frame with 7 residues subsequently of aminoterminal Met and beta galactosidase, thereby the preparation hybrid protein, pIN carrier (Van Heeke, G.andS.M.Schuster (1989) J.Biol.Chem.264:5503-5509) etc.(Promega, Madison Wis.) also can be used for expressing allogenic polypeptide as the fusion that has glutathione s-transferase (GST) the pGEX carrier.Usually, described fusion is soluble, and can have wash-out under the free glutathione afterwards by being adsorbed to glutathione-sepharose 4B, thus easily from cell lysis purifying come out.The albumen for preparing in the described system can be designed to comprise heparin, fibrin ferment or factor XA proteinase cutting site, so that clone's desired polypeptides can optionally partly discharge from GST.

In saccharomyces cerevisiae (Saccharomyces cerevisiae), can use the many carriers that contain composing type or inducible promoter, for example the α factor, alcohol oxidase, and PGH.Relevant summary is referring to Ausubel (the same) and Grant et al. (1987; Methods Enzymol.153:516-544).

In the situation of using plant expression vector, the expression of the sequence of coding Gpr100 GPCR can be by many promoters driven.For example, can use viral promotors separately,, or can be used in combination (Takamatsu, N. (1987) EMBO are J.6:307-311.) with ω targeting sequencing from TMV as 35S and the 19S promoter of CaMV.Perhaps, can use plant promoter, as the small subunit of RUBISCO, or the heat shock promoter (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.CellDiffer.17:85-105.).These constructs can import in the vegetable cell by the transfection that direct DNA transforms or pathogen mediates.Described technology in common available summary, describe (referring to, for example, Hobbs, S.or Murry, L.E.in McGraw Hill Yearbook of Science and Technology (1992) McGraw Hill, New York, N.Y.; Pp.191-196.).

The insect system also can be used to express Gpr100 GPCR.For example, in a kind of described system, autographa california (Autographa californica) nuclear polyhedrosis virus (AcNPV) is used as carrier with expression alien gene in fall army worm (Spodoptera frugiperda) cell or cabbage looper (Trichoplusia) larva.The sequence of coding Gpr100 GPCR can be cloned into the nonessential region of virus, polyhedron gene for example, and be subjected to the regulation and control of polyhedrin promoter.The successful insertion of Gpr100 GPCR will make the polyhedron gene inactivation, and produce the recombinant virus that lacks capsid protein.Afterwards, this recombinant virus can be used for infecting fall army worm cell or the cabbage looper larva that for example wherein can express Gpr100 GPCR.(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.As under the situation of expression vector, the sequence of coding Gpr100 GPCR can be connected to the adenovirus of being made up of late promoter and tripartite leader[and transcribe/translate in the compound in adenovirus.Insert virus genomic nonessential E1 or E3 district, can obtain in infected host cell, to express the live virus (Logan, J.and T.Shenk (1984) Proc.Natl.Acad.Sci.81:3655-3659.) of Gpr100 GPCR.And transcriptional enhancer as Rous sarcoma virus (RSV) enhancer, can be used for improving the expression in mammalian host cell.

Therefore, for example the Gpr100 acceptor can be expressed in human embryo kidney (HEK) 293 (HEK293) cell or adherent dhffCHO cell.Express maximization in order to make, before inserting pCDN or pCDNA3 carrier, usually will be whole 5 ' remove from receptor cdna with 3 ' untranslated district (UTR).Pass through the liposome transfection cell with independent receptor cdna, and under 400mg/ml G418, select.After selecting for 3 weeks, choose single clone and expansion and be used for further analysis.With the HEK293 of the independent transfection of carrier or Chinese hamster ovary celI as negative control.Express the clone of independent acceptor for separating stable, choose about 24 clones usually, and analyze with the Northern engram analysis.Receptor mrna usually can be in the G418-resistance clone of being analyzed about 50% in detect.

Human artificial chromosome (HAC) also can be used to transmit the bigger dna fragmentation of dna fragmentation than comprising and express in plasmid.Be therapeutic purposes, make up the HAC of about 6kb to 10Mb, and transmit by conventional transmission method (liposome, polycation amino polymer or carrier).

Also but end user's artificial chromosome (HAC) transmits than the big dna fragmentation of dna fragmentation that can comprise and express in plasmid.For making up about 6kb, the purpose for the treatment of transmits to the HAC of 10Mb and by conventional carrying method (liposome, polycation amino polymer or vesicle).

Also available special start signal translates more efficiently the sequence of coding Gpr100 GPCR.Described signal comprises ATG initiation codon and contiguous sequence.When the sequence of coding Gpr100 GPCR and initiation codon thereof and upstream sequence are inserted into suitable expression vector, can no longer need additional transcription or translation control signal.But, under the situation of only inserting coded sequence or its fragment, should provide the external source translational control that comprises ATG initiation codon signal.And initiation codon should be arranged in correct reading frame, to guarantee the translation of whole embolus.External source translation element and initiation codon can have multiple source, no matter are natural or synthetic.By comprising the enhancer that is suitable for used concrete cell system, for example described in the document, can improve the efficient of expression.(Scharf，D.et al.(1994)Results Probl.Cell Differ.20：125-162.)

In addition, can express, or select host cell strain with the ability that the mode of needs is processed expressed protein according to regulating insetion sequence.The described modification of polypeptide includes but not limited to, acetylation, carboxylation, glycosylation, phosphorylation, fatization and acidylate.Processing also can be used for promoting correct insertion, folding and/or function after the translation that " preceding former " form of protein is cut.Different host cells with peculiar mechanism of mechanism and translation back activity in the specific cells (for example, CHO, HeLa, MDCK, HEK293 and WI38) can be from American type culture collection (ATCC, Bethesda, Md.) obtain, and can be selected to guarantee the correct modification and the processing of foreign protein.

Extended high rate amount for recombinant protein is produced, and stable expression is preferred.For example, but clone that can stably express Gpr100 GPCR can transform with the expression vector that contains virus replication starting point and/or endogenous expression element and selectable marker gene (on identical or the carrier that separates).After importing carrier, before transferring to selective medium, can allow cell in enriched medium, to grow about 1 to 2 day.The purpose of selected marker is the resistance of giving selecting, and it exists and has allowed successful expression and import the growth and the recovery of the cell of sequence.The resistance clone of the cell of stable conversion can use the tissue culture technique that is fit to this cell type to breed.

Any amount of selective system can be used to reclaim cell transformed system.These include but not limited to, herpes simplex virus thymidine kinase gene (Wigler, M.et al. (1977) Cell 11:223-32) and adenine phosphoribosyl transferase gene (Lowy, I.et al. (1980) Cell 22:817-23), it can be respectively applied for tk-or apr-cell.Antimetabolite, microbiotic or Herbicid resistant also can be used as the basis of selection.For example, dhfr gives the resistance to methotrexate (MTX) (Wigler, M.et al. (1980) Proc.Natl.Acad.Sci.77:3567-70); Npt gives the resistance of aminoglycosides neomycin and G-418 (Colbere-Garapin, F.et al (1981) J.Mol.Biol.150:1-14); And als or pat give the resistance (Murry, the same) to chlorine sulphur grand (chlorsulfuron) and careless ammonium phosphine (phosphinotricin) transacetylase respectively.Additional selected gene has had description, trpB for example, its permissive cell utilize indoles to replace tryptophane, or hisD, its permissive cell utilizes histinol to replace histidine (Hartman, S.C.and R.C.Mulligan (1988) Proc.Natl.Acad.Sci.85:8047-51.).Recently, the use of witness marking comes into vogue, and these marks are anthocyanin (anthocyanin), beta-glucuronidase and substrate GUS thereof for example, and luciferase and substrate fluorescein thereof.These marks not only can be used for identifying transformant, also can be used to the instantaneous or stable protein expression amount of (attributeable) due to the quantitative specific support system.(Rhodes，C.A.et al.(1995)Methods Mol.Biol.55：121-131.)

Although the existence that marker gene is expressed/shortage hint genes of interest also exists, the existence of this gene and expression may need to confirm.For example, if the sequence of coding Gpr100 GPCR is inserted in the marker gene sequence, the transformant that contains the sequence of coding Gpr100 GPCR can be identified by the shortage of marker gene function.Perhaps, marker gene can be under the control of single promoter be arranged in series with the sequence of coding Gpr100GPCR.Marker gene responds the expression of inducing or selecting and has also shown the polyphone expression of gene usually.

Perhaps, contain the nucleotide sequence of coding Gpr100 GPCR and the host cell of expression Gpr100 GPCR, can identify by several different methods well known by persons skilled in the art.These methods include but not limited to, DNA-DNA or DNA-RNA hybridization, and protein biological detection or immunoassay technology, and it comprises and is used for detecting and/or the technology based on film, solution or chip of quantitative nucleic acid or protein sequence.

The existence of the polynucleotide sequence of coding Gpr100 GPCR can be by utilizing probe or fragment or coding Gpr100 GPCR the DNA-DNA of polynucleotide passage or DNA-RNA hybridization or amplification detect.Relate to use based on the detection of nucleic acid amplification and detect the DNA that contains coding Gpr100 GPCR or the transformant of RNA based on the oligonucleotides of the sequence of coding Gpr100 GPCR or oligomer.

It is well known by persons skilled in the art that utilization detects and measure the several different methods that Gpr100GPCR expresses to the special polyclone of this protein or monoclonal antibody.The example of described technology comprises enzyme linked immunosorbent assay (ELISA) (ELISA), radiommunoassay (RIA) and fluorescence activated cell sorting (FACS).Utilize with Gpr100 GPCR on the dibit point that carries out of the monoclonal antibody that reacts of two non-interference epi-positions, be preferred based on monoclonal immunoassays, but also can use the competitive binding assay method.These and other determination methods have a detailed description in the art, Hampton for example, and R.etal (1990; Serological Methods, a Laboratory Manual, Section IV, APS Press, St Paul, Minn.) and Maddox, D.E.et al. (1983; J.Exp.Med.158:1211-1216).

Multiple label and coupling technology are well known by persons skilled in the art, and can be used for various nucleic acid and amino acid analysis.The preparation mark hybridization or the PCR probe detects and the method for the polynucleotide correlated series of the Gpr100 GPCR that encodes comprises the pcr amplification of oligomerization mark (oligolabeling), nick translation, end mark or usage flag nucleotide.Perhaps, the sequence of coding Gpr100 GPCR, or its any fragment can be cloned into the carrier that is used to prepare the mRNA probe.Described carrier is known in the art, is that commercialization provides, and by adding suitable R NA polymerase, for example the nucleotide of T7, T3 or SP6 and mark is used in the vitro synthesized RNA probe.These methods can be carried out with the kit that multiple commercialization provides, for example by Pharmacia; Upjohn (Kalamazoo, Mich.), GE Healthcare (UK) and U.S.Biochemical Corp. (Cleveland, the kit that Ohio) provides.Suitable reporter molecules that is advantageously used in detecting or label comprise radioactive nuclide, enzyme, fluorescence, chemiluminescent or developer, and substrate, co-factor, inhibitor, magnetic-particle etc.

Nucleotide sequence transformed host cells with coding Gpr100 GPCR can be cultivated under the condition that is suitable for protein expression and reclaims from cell culture.Protein by the transformant preparation can be positioned on the cell membrane, secretes or be included in cell according to the sequence and/or the carrier that use.As skilled in the art to understand, the expression vector that contains the polynucleotide of coding Gpr100 GPCR can be designed to comprise and instruct the burst of Gpr100 GPCR by the cell membrane secretion of protokaryon or eucaryon.Other constructs can be used to the sequence of coding Gpr100 GPCR is connected with the nucleotide sequence that coding is beneficial to the polypeptide structure territory of soluble protein purifying.The domain of described promotion purifying includes but not limited to, metal-chelate is closed peptide, as allow the histidine-tryptophane assembly that on fixing metal, carries out purifying, allow the a-protein domain of purifying on fixing immunoglobulin (Ig), and domain (the Immunex Corp. that is used for FLAGS extension/protein affinity purification system, Seattle, Wash.).Comprise resectable catenation sequence between purification structure territory and Gpr100GPCR coded sequence, for example (Calif.) special sequence can promote purifying for Invitrogen, San Diego to factor XA or enterokinase.A kind of described expression vector can make and contain Gpr100 GPCR and be coded in thioredoxin or the expressing fusion protein of the nucleic acid of 6 histidine residues that the enterokinase cleavage site is preceding.Histidine residues is beneficial to the purifying (IMIAC on fixing metallic ion affinity chromatography; Porath describes among J.et al. (1992) Prot.Exp.Purif.3:263-281), and the enterokinase cleavage site provides from the means of fusion purifying Gpr100 GPCR.Argumentation to the carrier that contains fusion is seen Kroll, D.J.et al. (1993; DNA Cell Biol.12:441-453).

The fragment of Gpr100 GPCR not only can be by reorganization preparation, and can synthesize by the direct peptide that utilizes solid phase technique (Merrifield J. (1963) J.Am.Chem.Soc.85:2149-2154.) and prepare.Protein synthesis can be by the artificial technology or is carried out automatically.Automatically synthetic can passing through for example uses Applied Biosystems 431A peptide synthesizer (Perkin Elmer) to realize.The multiple fragment of Gpr100 GPCR can be synthesized respectively, afterwards the molecule of combination results total length.

Biology sensor

Gpr100 polypeptide, nucleic acid, probe, antibody, expression vector and part can be used as (and can be used for producing) biology sensor.

According to Aizawa (1988), AnaL Chem.Symp.17:683, biology sensor is defined as, the unique combination that is used for the acceptor of molecular recognition, for example, has the selective layer of sessile antibody or acceptor such as Gpr100 g protein coupled receptor and be used to transmit the transmitter of measured value.One group of such biology sensor will detect the variation that causes owing to the interaction of this receptor and surrounding medium on the optical characteristics of superficial layer.In described technology, can mention elliptic polarization and surface plasma resonance art especially.The biology sensor that is combined with Gpr100 can be used for detecting the existence or the level of Gpr100 part, nucleotide for example, for example purine or purine analogue, or the analog of these parts.The structure of described biology sensor is well known in the art.

Therefore the clone of expressing the Gpr100 acceptor can be by means of [3H] phosphoinositide of acceptor promotion or other second messengers' formation, as reporting system (Watt et al., 1998, the J Biol Chem May 29 of detector ligand such as ATP; 273 (22): 14053-8).The receptor-ligand biology sensor is also at Hoffinan et al., and 2000, Proc Natl Acad Sci USA Oct 10; 97 (21): description is arranged among the 11215-20.Comprise the optics of Gpr100 and other biological sensor and also can be used to detect level or existence with G albumen and other protein interactions, as for example Figler et al, 1997, BiochefsaistfyDec 23; 36 (51): 16288-99 and Sarrio et al., 2000, Mol Cell Biol 2000 Jul; 20 (14): 5164-74) described.The sensor unit of biology sensor is at for example US5, describes in 492,840.

Screening is analyzed

Gpr100 GPCR polypeptide comprises homologue, variant and derivant, no matter is natural or reorganization, all can be applicable to bind receptor and activation (activator) or suppresses in the screening technique of compound of (inhibitor) Gpr100 activation.Therefore, the Gpr100 polypeptide also can be used for assessing micromolecule substrate and part for example, the combination in cell, not celliferous goods, chemical library and the natural product mixture.These substrates and part can be natural substrate and parts, maybe can be structural or functional analogies.Referring to Coligan et al., Current Protocols in Immunology 1 (2): Chapter 5 (1991).

Gpr100 GPCR polypeptide determines a lot of biological functions, comprises a lot of pathology.Therefore, wish to find the compound and the medicine that stimulate Gpr100 GPCR on the one hand and suppress Gpr100 GPCR function on the other hand.Usually, activator and antagonist are used for treatment and the prevention purpose as the state of an illness of Gpr100 relevant disease.To may with the appropriate design of the interactional candidate compound of Gpr100 gpcr protein can be based on the structural research of the molecular shape of polypeptide.A kind ofly determine which site and the method for specific other protein interactions are physical arrangement mensuration, for example, X-radiocrystallography or two dimensional NMR techniques.This can provide the guidance which amino acid residue to form the molecule contact area for.About the detailed description of protein structure mensuration, referring to for example Blundell and Johnson (1976) Protein Crystallography, Academic Press, New York.

The another kind of appropriate design is selected to utilize screening technique, described method to relate to the common preparation of suitable cell, and described cell is expressed the Gpr100 receptor polypeptides in its surface.Described cell comprises from animal, yeast, fruit bat or colibacillary cell.Afterwards the cell of the expressed receptor cell membrane of expressed acceptor (or contain) is contacted with tested compound with observe in conjunction with or the stimulation or the inhibition of functional response.For example, available Gpr100 mRNA or polypeptide injection xenopus leavis oocytes, and utilize voltage clamp to measure by contacting the electric current that tested compound is induced, see other part more detailed descriptions.

In addition, little physiological measurements (microphysiometric) analysis can be used for analyzing the Gpr100 receptor active.The activation of multiple second messenger system causes that small amount of acid extrudes from cell.The acid that forms mainly is the result of the metabolic activity of enhancing, and described metabolic activity provides energy required for signal conductive process in the cell.The change of cell peripheral medium pH is very little but can (Molecular Devices Ltd., Menlo Park Calif.) detect by the little physiology meter of CYTOSENSOR (microphysiometer) for example.Therefore CYTOSENSOR can detect and the acceptor of the interior signal transduction path coupling of the cell that utilizes energy such as the activation of Gpr100 g protein coupled receptor.

Except that detecting respectively each candidate compound, can produce and screen library or storehouse (bank) of candidate ligand easily with the Gpr100 acceptor.Therefore, for example, assembled and surpass 200 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 by seven of people; Naturally occurring compound, it can be the activator that is estimated as the not certified as yet people 7TM acceptor of its mammal homologue, nonmammalian, biologically active peptide; And non-natural exists but activates the compound of 7TM acceptor with unknown native ligand.As other parts functional selection (that is, calcium, cAMP, little physiology meter, egg mother cell electrophysiology etc. are seen other parts) and binding analysis in greater detail, this storehouse is used to screen the acceptor of known ligand.But, still there is a large amount of mammalian receptors, it does not have the activation part (activator) or the passivation part (antagonist) of homology so far.Therefore the activation part of these acceptors may not be included in the part storehouse of having identified up to now.Therefore, also can carry out functional screening (using calcium, cAMP, little physiology meter, egg mother cell electricity physiology method etc., functional screening) to the Gpr100 acceptor and identify natural part at tissue extract.The extract that produces positive functional response can continue by inferior classification, and its fraction is as described herein to be analyzed, up to separating and identifying the activation part.

The 7TM acceptor of expressing in HEK 293 cells has shown to stimulate with the activation of PLC and calcium migration and/or cAMP or is suppressed at coupling on the function.Therefore, a kind of triage techniques is included in the system that the outer pH of born of the same parents that measurement causes by receptor activation or intracellular Ca2+ change and utilizes the cell (for example, the xenopus leavis oocytes of transfection, CHO or HEK293 cell) of expressing Gpr100 GPCR acceptor.In this technology, compound can with the cells contacting of expressing the Gpr100 receptor polypeptides.Whether measure the second messenger afterwards and reply, for example, signal transduction, pH change or the change of calcium level, activate or suppress this receptor to determine potential compound.

In described experiment, the basic calcium level of observing HEK 293 cells in acceptor transfection or the vehicle Control cell is in normal 100nM within the 200nM scope.Express the HEK293 cell of Gpr100 GPCR or reorganization Gpr100 GPCR and go up sample with fura2, and in one day, to surpassing the calcium migration of 150 selected parts or tissue/cell extract assessment agonist induction.Similarly, utilize the cAMP quantitative determination process assessment of standard to stimulation or the inhibition of expressing Gpr100 GPCR or the HEK 293 cell cAMP of the Gpr100 GPCR that recombinates produce.Whether detection presents the activator that calcium is instantaneous or cAMP fluctuates in the vehicle Control cell, reply exclusive by the transfectional cell of expressed receptor to determine this.

Another kind method relates to by the inhibition of determining the accumulation of receptor-mediated cAMP of Gpr100 and/or adenyl cyclase or stimulates screens acceptor inhibitor.Described method comprises with Gpr100 acceptor transfecting eukaryotic cells with expressed receptor on cell surface.This cell contacts potential antagonist when having acceptor afterwards.Measure the amount of cAMP accumulation subsequently.If potential antagonist bind receptor, and therefore suppress receptors bind, receptor-mediated cAMP or adenylate cyclase enzyme level, then active the reduction or raising.

In preferred embodiments, this screening uses the detection of intracellular calcium concentration variation to screen activator and the antagonist of Gpr100.Particularly, we disclose a kind of method, wherein in the cell that the antagonist of Gpr100 reduces, weakens or block ligand is induced, the calcium in the preferred suitable cells transfected discharges.Preferably, when having the antagonist of Gpr100, the raising of intracellular Ca2+ level reduced by 10%, 20%, 30%, 40%, 50%, 60%, 70% or more than.Preferably, when having the antagonist of Gpr100, intracellular Ca2+ discharge reduced 1mM, 2mM, 3mM, 4mM, 5mM, 10mM, 15mM, 25mM, 35mM, 45mM, 60mM, 70mM or more than.

We further disclose a kind of method, and wherein the activator of Gpr100 improves the concentration of intracellular Ca2+ in the suitable cells transfected.Preferably, when having the activator of Gpr100, electricity lead improved 10%, 20%, 30%, 40%, 50%, 60%, 70% or more than.Preferably, when having the activator of Gpr100, electricity lead improved 1mM, 2mM, 3mM, 4mM, 5mM, 10mM, 15mM, 25mM, 35mM, 45mM, 60mM, 70mM or more than.

The another kind of method that is used to detect Gpr100 receptor stimulating agent or antagonist is for based on U.S. Patent No. 5,482, and the yeast of technology described in 835 is hereby incorporated by.

When candidate compound is a protein, especially when antibody or peptide, can utilize the library of display technique of bacteriophage screening candidate compound.Phage display is a molecular screening scheme of utilizing recombinant phage.This technology relates to the genetic transformation bacteriophage of coding from the compound in candidate compound library, so that each bacteriophage or phasmid are expressed specific candidate compound.Bacteriophage (preferably being connected on the solid support) through transforming is expressed suitable candidate compound, and it is illustrated on the bacteriophage capsid.Can be in conjunction with the particular candidate compound of Gpr100 polypeptide or peptide by based on the interactional selection strategy of affinity and enrichment.Characterize successful candidate agent subsequently.Phage display has more advantage than the affinity ligands triage techniques of standard.Phage surface is showed candidate agent with 3-d modelling, has more closely reflected its naturally occurring conformation.This provides, and to be used to screen purpose more special and have a more combination of high-affinity.

The another kind of method utilization in the screening compounds library eucaryon or the prokaryotic host cell of the recombinant DNA molecules stable conversion of expressing library of compounds.Described cell no matter be survival or fixing form, can be used in the combination-counter pair analysis of standard.Also referring to Parce et al. (1989) Science246:243-247; With Owicki et al. (1990) Proc.Nat ' 1 Acad.Sci.USA 87; 4007-4011, it has described the sensitive method that detects cell effect.Competitive assay is particularly useful, wherein express the cell of library of compounds and the labelled antibody of the known Gpr100 of combination polypeptide, 125I-antibody for example, with contacted or incubation by test agent, described by test agent candidate compound for example, the binding affinity of described candidate compound and binding compositions is measured.Combination of isolated polypeptide subsequently and free mark binding partners are with the assessment combination degree.Be inversely proportional to by the amount of the binding capacity of test agent with the labelled antibody that combines polypeptide.

Any can both being used in the multiple technologies separates the binding partners with free of combination to assess the degree of combination.This separating step generally includes following operation steps, for example is attached to filter, and washing is attached to plastics then, washing, or centrifuge cell film afterwards.

Also having another kind of method is to utilize soluble, not purifying or soluble purified polypeptide or peptide, and described polypeptide or peptide for example extract from the eucaryon that transforms or prokaryotic host cell.This allows to carry out advantage and is the specificity, automatic capability and the drug test that improve high-throughout " molecule " binding analysis.

The another kind of technology of candidate compound screening relates to the method that the high flux screening noval chemical compound is provided, described noval chemical compound for example has suitable binding affinity to the Gpr100 polypeptide, and describes in detail in disclosed international patent application no WO 84/03564 on September 13rd, 1984 (Commonwealth Serum Labs.).At first, a large amount of different tested compounds of little peptide are at solid-phase matrix, and are for example, synthetic on plastics pin (pin) or some other suitable surfaces; Referring to Fodor et al. (1991).Subsequently, reaction of the Gpr100 polypeptide of whole pin and dissolving and washing.Next procedure comprises the polypeptide that detects combination.So identify and the interactional compound of polypeptid specificity.

The part binding analysis provides the pharmacological direct method of definite acceptor and has been applicable to high throughput format.But being high specific activity (50-2000Ci/mmol), the receptors ligand radioactive label of purifying is used in conjunction with research.Determine that subsequently radiolabeled process can not weaken the activity of part to its acceptor.Optimize the analysis condition of damping fluid, ion, pH and other correctivess such as nucleotide, with the available signal-to-noise ratio in the acceptor source of setting up film and whole cell.Analyze for these, specific receptor deducts the radioactivity of measuring in conjunction with being defined as total relevant radioactivity when having excessive unlabelled competitive part.If possible, use more than one competitive part to limit residual non-specific binding.

This analysis can be tested the combination of candidate compound simply, wherein with the adhering to by means of the label directly or indirectly relevant with this candidate compound or detected in relating to the analysis of competing with the mark competitor of the cell that carries acceptor.Further, utilize the detection system that the cell that carries acceptor on its surface is fit to, these analyses can be tested this candidate compound and whether cause the signal that produced by receptor activation.Usually when having known activator, measure the inhibitor of activation, and observe the effect of activator to activating when having candidate compound.

In addition, the solution that this analysis can only comprise the following steps: to mix candidate compound and comprise the Gpr100GPCR polypeptide is measured the activity of Gpr100 GPCR in the potpourri to form potpourri, and the Gpr100 GPCR activity of potpourri is compared with reference material.

The antibody of Gpr100 GPCR cDNA, protein and this protein also can be used for the analysis of the effect of the generation of Gpr100 GPCR mRNA and protein in the compound pair cell that configuration detection adds.For example, utilize monoclonal and polyclonal antibody, can make up ELISA secretion or the cell related levels that measures the Gpr100 gpcr protein by standard method known in the art, and this can be used for finding to suppress or to strengthen the reagent (also being called antagonist or activator) for preparing Gpr100 GPCR from the cell or tissue of proper handling.The standard method that is used to screen analysis is well known in the art.

The example of potential Gpr100 GPCR antagonist comprise antibody or, sometimes comprise nucleotide and analog thereof, comprise purine and purine analogue, oligonucleotides or protein, the part of itself and Gpr100 GPCR is closely related, for example part fragment, or bind receptor but do not excite the micromolecule of replying, making that the activity of acceptor is hindered.

This paper therefore also provide can specificity in conjunction with the compound of Gpr100 polypeptide and/or peptide.

Term " compound " refers to (naturally occurring or synthetic) chemical compound, as biomacromolecule (for example, nucleic acid, protein, non-peptide or organic molecule), or from the extract of the biomaterial of for example bacterium, plant, fungi or animal (especially mammal) cell or tissue preparation, and inorganic elements or molecule.Preferred this compound is an antibody.

Can be packaged in the screening reagent box for carrying out the essential material of this screening.Described screening reagent box can be used for identifying the activator, antagonist, part, acceptor, substrate, enzyme of Gpr100 GPCR polypeptide etc., or reduces or improve the compound that Gpr100 GPCR polypeptide produces.This screening reagent box comprises: (a) Gpr100 GPCR polypeptide; (b) recombinant cell of expression Gpr100 GPCR polypeptide; (c) cell membrane of expression Gpr100 GPCR polypeptide; Or (d) antibody of anti-Gpr100 GPCR polypeptide.This screening reagent box optionally comprises operation instructions.

Transgenic animals

This paper further comprises and normal expression specific energy or the Gpr100 GPCR of reorganization or transgenic animals of homologue, variant or derivant natural with the horizontal expression that improves or reduce mutually.Also comprise sudden change, comprise the result of Gpr100 gene delection and the transgenic animals (" Gpr100 knocks out ") of non-expressive function property Gpr100 acceptor as one or more losses of function.Preferably, described transgenic animals are non-human mammal, as pig, sheep or rodent.Most preferably these transgenic animals are mouse or rat.Described transgenic animals can be used in the screening technique identifying activator and/or the antagonist of Gpr100 GPCR, and detect its effectiveness as disease treatment in the body.

For example, defective transgenic animals can be used in the determination method to identify activator and/or the antagonist of Gpr100 GPCR in Gpr100 GPCR generation through transforming.A kind of determination method is designed to assess potential medicine (candidate ligand or compound) to determine whether it produces physiological reaction under shortage Gpr100 GPCR acceptor.This can be by giving medicament administration above-mentioned transgenic animals, and the specific reaction of measuring described animal subsequently realizes.Though can be in this determination method any physiological parameter, preferred reaction comprises following one or more: the variation of disease resistance; The change of inflammatory reaction; The change of tumor susceptibility: the variation of blood pressure; Neovascularization; The variation of dietary 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.

The tissue that is derived from the Gpr100 knock-out animal can be used for receptors bind and measures to determine that whether potential medicine (candidate ligand or compound) is in conjunction with the Gpr100 acceptor.Described mensuration can be by obtaining the first acceptor goods and obtain the second acceptor goods and carry out from the known Gpr100 part or the source of compound in conjunction with any evaluation from being transformed into transgenic animals that the Gpr100 acceptor produces defective.Usually, except that the source that obtains, the first and second acceptor goods are all similar in all fields.For example, if be used for measuring, be used as the source of the second acceptor goods from the comparable brain tissue of normal (wild type) animal from the brain tissue (as mentioned with described below) of transgenic animals.Individually and in the presence of candidate ligand or compound, each acceptor goods is with known part incubation in conjunction with the Gpr100 acceptor.Preferably, candidate ligand or compound are measured with several different concentration.

For the first and second acceptor goods, measure the combination degree that substitutes known ligand with test compounds.The tissue that is derived from transgenic animals can be directly used in mensuration, perhaps can process this tissue to separate film or the memebrane protein that itself is used to measure.Preferred transgenic animals are mouse.Can use any method tagged ligand that is suitable in conjunction with measuring.This includes, without being limited to, radioactive, enzymatic, fluorescence or chemiluminescent labeling (and other labelling technique that above describes in further detail).

In addition, wild type animal that can be by candidate compound etc. being given expressive function Gpr100 and identify that it presents and reduces or eliminate the animal that the Gpr100 function of receptors is expressed relevant any phenotypic characteristic, and the antagonist of evaluation Gpr100 GPCR acceptor.

The detailed method that produces the non-human transgenic animal hereinafter has been described in further detail.Genetically modified gene construct can import animal reproduction system to produce genetically modified mammal.For example, the construct of one or several copy can be integrated into the genome of mammal embryo by the transgenic technology of standard.

In illustrative embodiment, genetically modified non-human animal produces by the system genitale that transgenosis is imported the non-human animal.Embryo target cell in different developmental phases can be used for importing transgenosis.Stage of development according to the embryo target cell is used diverse ways.At visible good pronucleus and good reproduction adaptability among healthy, good embryo's productive rate, the embryo generally, select the specific kind system of any animal.In addition, haplotype is important factor.

Transgenosis imports the embryo and can realize by any method known in the art, for example, and microinjection, electroporation or lipofection.For example, can by with the construct microinjection in the pronucleus of mammalian zygote and Gpr100 acceptor transgenosis is imported mammal, thereby cause the construct of one or more copies to remain in the developmental mammiferous cell.After transgenic constructs imported embryonated egg, this ovum can be in the time of in vitro culture different length, or replant into the replace-conceive host, or both all carry out.Also can carry out in vitro culture to ripe.Usual method is according to species, at the about 1-7 of extracorporeal culturing embryo days, then it is replanted into the replace-conceive host.

Can test the existence of this construct among the embryo offspring of transgeneic procedure by Southern engram analysis tissue fragment.If the external source of one or more copies clone construct keeps stably being incorporated in the genome of this transgenic embryo, then might set up the lasting transgene mammal system of carrying the construct that adds with transgenic method.

The mammiferous nest son that transgenosis changes can be measured this construct mixing in offspring's genome at the birth post analysis.Preferably, this is measured by making corresponding to the dna sequence dna of coding desired recombinant protein matter product or the probe hybridization of its sections and realizes to the chromosomal material from the offspring.Those mammiferous offsprings that are found in the construct that comprises at least one copy in its genome grow to maturation gradually.

For the purpose of this paper, zygote is the formation of diploid cell basically, and described diploid cell can the complete biosome of bud into.Usually, zygote is made up of the ovum that comprises nuclear, and the natural or artificially of described nuclear forms by the fusion from two haploid nuclears of one or more gamete.Therefore, the nuclear of gamete must be natural compatible, promptly produces to experience the zygote that viability is arranged that differentiation and bud into have the function biosome.Usually, euploid zygote is preferred.If obtain the zygote of aneuploid, then with regard to the euploid number of the biosome of originating with regard to arbitrary gamete, the variation of chromosomal number will can be above 1.

Except that similar biological factor, physical factor is also controlled the amount (for example, volume) of the exogenous genetic material of the inhereditary material that can add syncaryon or add a part that forms syncaryon.If do not remove inhereditary material, then the amount of the exogenous genetic material that can add is subjected to not by the also absorbed quantitative limitation of physical damage.Usually, the volume of the exogenous genetic material of insertion can not surpass about 10 skin liters.The physical influence that adds needn't too big viability with this zygote of physical damage.The number of dna sequence dna and the restriction of the biology of kind will change according to the function of specific zygote and exogenous genetic material, and it will be apparent to those skilled in the art that, because the inhereditary material of resulting zygote, comprise exogenous genetic material, must be biologically can be initial and keep this zygote differentiation and development and become the biosome that function is arranged.

The copy number that adds the transgenic constructs of embryonated egg depends on the total amount of the exogenous genetic material of adding, and will be the quantity that genetic transformation is taken place.It is necessary having only a copy in theory; Yet, use many copies usually, for example 1,000-20, the transgenic constructs of 000 copy has function to guarantee a copy.The exogenous DNA array of each insertion has an above function, and to copy the phenotypic expression that strengthens exogenous DNA array often be favourable.

Can utilize and allow that exogenous genetic material adds any technology of nuclear genetic material, as long as its pair cell, nuclear membrane or other already present cells or genetic structure are not harmful to.The inhereditary material of external source preferably inserts the nuclear genetic material by microinjection.Cell and cyto-architectural microinjection are known in the art and commonly used.

Reimplantation utilizes the method for standard to finish.Usually, anaesthetize the replace-conceive host, and the embryo is inserted fallopian tubal.The number of implanting the embryo of specific host will change according to species, but common and the natural generation offspring's of these species number has comparability.

Can be by this genetically modified existence and/or expression in any suitable method screening replace-conceive host's the transgenic progeny.Screening utilizes with genetically modified probe to the small part complementation and finishes often by Southern trace or Northern engram analysis.Utilization resists the Western engram analysis by the antibody of the protein of transgenes encoding to can be used as the alternative or additional method that this transgene product of screening exists.Normally, DNA analyzes transgenosis from the afterbody tissue preparation and by Southern analysis or PCR.Optional, utilize Southern to analyze or PCR tests and is considered to express in this genetically modified tissue or the cell this with highest level and genetically modifiedly exists and express, although any tissue or cell type can be used for this analysis.

Be used to assess the optional or additional method that transgenosis exists and include but not limited to suitable biochemical measurement, for example tissue staining of enzyme and/or immunologic assay, particular marker or enzymatic activity, flow cytometry etc.The analysis of blood also can be used for detecting the existence of transgene product in the blood, and is used to assess the influence of this transgenosis to the level of various types of haemocytes and other blood constituents.

The offspring of transgenic animals can pass through render transgenic animal and suitable spouse's mating, or obtains by ovum and/or the in vitro fertilization of sperm that obtains from transgenic animals.In the time will carrying out mating with the spouse, this spouse may or may be not genetically modified and/or knocks out; When its when being genetically modified, can comprise identical or different transgenosis, or both.Optional, the spouse can be a parental line.When using when in vitro fertilization, the embryo of fertilization can implanted replace-conceive host or in vitro culture, or both.Use any method, can use aforesaid method, or other suitable methods are assessed genetically modified existence among the offspring.

The inhereditary material that will comprise external source according to the transgenic animals of method generation described herein.As mentioned above, in certain embodiments, the inhereditary material of external source is the dna sequence dna that can cause producing Gpr100 GPCR acceptor.In addition, in described embodiment, this sequence will be attached to transcriptional regulatory element, for example preferably allow this transgene product expression promoter in specific cell type.

Retroviral infection also can be used for transgenosis is imported the non-human animal.But developmental non-human embryo in vitro culture is to the blastocyst stage.During this time, blastomere can be the target (Jaenich, R. (1976) PNAS 73:1260-1264) of retroviral infection.Effective infection to blastomere can obtain (Manipulating the Mouse Embryo, Hogan eds. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1986) by enzymatic treatment to remove oolemma.Be used to import retrovirus (Jahner et al. (1985) PNAS82:6927-6931 that genetically modified virus carrier system normally carries genetically modified replication defective; Van der Putten et al. (1985) PNAS 82:6148-6152).Transfection realizes (Van der Putten, supra easily and effectively by cultivate blastomere on the cell monolayer of the cell that produces virus; Stewart et al. (1987) EMBOJ.6:383-388).Perhaps, infection can be carried out in the stage after a while.The cell of virus or generation virus can be injected into blastocoele (Jahner et al. (1982) Nature 298:623-628).The major part person of foundation (founder) will be genetically modified chimera, take place in the cell subgroup (subset) that forms the transgenic nonhuman animal because only mix.In addition, this person of foundation can comprise genetically modified some retrovirus emboluss at genomic diverse location, and described embolus separates in the offspring usually.In addition, also may with transgenosis by second trimester of pregnancy (midgestation) embryo the intrauterine reverse transcription infect and to import system genitale (Jahner et al. (1982) is the same).

The 3rd class target cell that is used for the transgenosis importing is embryo's a stem cell (ES).The ES cell merges (Evans et al. (1981) Nature 292:154-156 from the PIE acquisition of in vitro culture and with the embryo; Bradley et al. (1984) Nature 309:255-258; Gossler et al. (1986) PNAS83:9065-9069; And Robertson et al. (1986) Nature 322:445-448).Transgenosis can import the ES cell effectively by the DNA transfection or by the transduction of retrovirus-mediated method.Can combine with blastocyst after the ES cell of Zhuan Huaing like this from the non-human animal.This ES celluar localization afterwards is in the embryo and to the system genitale of gained chimaeric animals make contributions (contribute).Summary is referring to Jaenisch, R. (1988) Science 240:1468-1474.

Inhuman transgenic animals also are provided, and wherein the characteristics of these transgenic animals are to have the Gpr100 gene of change, preferably as mentioned above, and as the model of Gpr100 function of receptors.The change of this gene is comprised the sudden change of disappearance or other losses of function, import the foreign gene of nucleotide sequence, import foreign gene from another species with target or random mutation, or its combination.These transgenic animals for this variation can be isozygoty or heterozygosis.Animal that derives thus and cell are useful to the biologically active agent of screening scalable Gpr100 function of receptors.This screening method is used in particular for measuring obesity, the specificity and the effect of the potential therapy of especially appetite inhibition, lipid metabolism.This animal is as the model of the effect of research Gpr100 acceptor in normal brain, heart, spleen and liver function.

Another aspect is to have the endogenous Gpr100 gene that function is destroyed, but also carries in its genome, and expresses the transgenic nonhuman animal of the coding allos Gpr100 albumen Gpr100 of another species (that is, from).Preferably, this animal is a mouse, and the Gpr100 of allos is people Gpr100.Animal that the available Gpr100 that chosen rebuilds or the clone that is derived from described animal are identified in the body and the reagent of vitro inhibition people Gpr100.For example, can when tested reagent exist and lack, will be applied to animal or clone by the stimulation of people Gpr100 inducement signal conduction, and can measure the reaction in animal or the clone.In the body or the reagent of vitro inhibition people Gpr100 can be according to the reacting phase ratio when lacking this reagent, reaction reduces and identifies when having this reagent.

The disclosure also provides the transgenic nonhuman animal (" Gpr100GPCR knock-out animal ") of Gpr100 GPCR defective.Described animal is preferably the result that endogenous Gpr100 gpcr gene group sequence is destroyed or lack for expressing the animal that reduces or do not have Gpr100 GPCR activity.Preferably, described animal is not expressed the GPCR activity.More preferably, this animal is not expressed the activity of Gpr100 GPCR shown in SEQ ID NO:3 or SEQ ID NO:5.Gpr100 GPCR knock-out animal can be by prepared in various methods known in the art, as hereinafter described in more detail.

Content of the present disclosure also comprises the nucleic acid construct that is used at the functional destruction of host cell Gpr100 gene.This nucleic acid construct comprises: a) nonhomologous replacing section; B) be positioned at first homologous region of non-homogeneous replacing section upstream, this first homologous region has and the same substantially nucleotide sequence of a Gpr100 gene order, and c) is positioned at second homologous region in non-homogeneous replacing section downstream, this second homologous region has and the same substantially nucleotide sequence of the 2nd Gpr100 gene order, and the 2nd Gpr100 gene order is positioned at the position in a Gpr100 gene order downstream in naturally occurring endogenous Gpr100 gene.In addition, when this nucleic acid molecules imported host cell, first and second homologous regions had the sufficient length of carrying out homologous recombination between the endogenous Gpr100 gene in nucleic acid construct and host cell.In an embodiment preferred, non-homogeneous replacing section comprises expresses report, preferably includes lacZ and the positive expression cassette of selecting, and preferably includes the neomycin phosphotransferase gene that effectively is connected with one or more controlling elements.

Preferably, the described first and second Gpr100 gene orders are derived from SEQ ID No.1, SEQ IDNo.2 or SEQ ID NO:4 or its homologue, variant or derivant.

Another aspect of the disclosure comprises the recombinant vector that mixes nucleic acid construct.Another aspect comprises and imports nucleic acid construct, thereby allows homologous recombination between the endogenous Gpr100 gene of this nucleic acid construct and host cell, the host cell that causes the function of endogenous Gpr100 gene to be destroyed.This host cell can be that normal expression is from liver, brain, spleen or heart or pluripotent cell, as the mammalian cell of the Gpr100 of mouse embryo stem cell.Imported nucleic acid construct and with the further growth of the embryonic stem cell of endogenous Gpr100 dna homolog reorganization, produced and had the transgenic nonhuman animal of going down to posterity and therefore its genome, carrying the cell of Gpr100 gene disruption from embryonic stem cell.Therefore can be chosen in the animal of carrying the Gpr100 gene disruption in its system genitale, and make its breeding in whole body cells and reproduction cell, have the animal of Gpr100 gene disruption with preparation.Therefore described mouse can breed to the Gpr100 gene disruption has homozygosity.

Vitro system can be used to identify can be in conjunction with the compound of Gpr100 acceptor gene product.Described compound can include but not limited to, the peptide of forming by D-and/or L-BR BR configuration amino acid (for example with the random peptide library form), phosphopeptide (for example with at random or the part degeneracy, direct phosphopeptide library form), antibody and little organic or inorganic molecule.Compounds identified for example can be used for regulating Gpr100 acceptor gene albumen, the activity of preferred mutant Gpr100 acceptor gene albumen; The biological function of performance Gpr100 acceptor gene albumen; Or screening destroys, and normal Gpr100 acceptor gene interacts or itself destroys described interactional compound.

Demonstration can further detect it in conjunction with the compound of specific Gpr100 acceptor gene product and cause ability from the biochemical reaction of Gpr100 acceptor gene albumen.The activator of expression product, antagonist and/or inhibitor can utilize determination method well known in the art to identify.

Antibody

For the purpose of this paper, unless stated otherwise, term " antibody " includes but not limited to, polyclone, monoclonal, chimeric, strand, Fab fragment and by the fragment of Fab expression library preparation.These fragments comprise fragment, Fv, F (ab ') and the F (ab ') in conjunction with active complete antibody of maintenance to the target material ₂Fragment, and single-chain antibody (scFv), fusion and other comprise the synthetic proteins of the antigen binding site of this antibody.Antibody and fragment thereof can be humanized antibodies, for example as described in the EP-A-239400.In addition, also can use antibody with complete people variable region (or its fragment), as U.S. Patent No. 5,545,807 and 6,075,181 is described.Neutralizing antibody promptly suppresses the bioactive antibody of described material amino acid sequence, especially is preferred for diagnosis and treatment.

Antibody can pass through standard technique, as preparing by immunity inoculation or use phage display library.

Can develop antibody with polypeptide or peptide by known technology.Described antibody capable specificity is in conjunction with Gpr100 gpcr protein or homologue, fragment etc.

Polyclonal if desired antibody, the available immunogenic composition immunity inoculation that comprises Gpr100 polypeptide or peptide of selected mammal (for example, mouse, rabbit, goat, horse etc.).According to host species, various adjuvants can be used to improve immune response.Described adjuvant includes but not limited to, Fu Shi, mineral salt gel for example lysolecithin, polyether polyhydroxy-compound (pluronic polyol) of aluminium hydroxide and surface reactive material for example, polyanion, peptide, fat liquor, keyhole limpet hemocyanin (keyhole limpethemocyanin) and dinitrophenol dinitrophenolate.BCG (Bacille Calmette-Guerin) and CBP (Corynebacteriumparvum) are the human adjuvant that comes in handy, if when the purifying amino acid sequence of described material is administered to the individuality that lacks immunity and defends with stimulating system, can use described adjuvant.

Collect to handle from the serum of immunity inoculation animal and according to known method.If comprise at the antibody that can comprise other antigens available from the serum of the polyclonal antibody of the epi-position of Gpr100 polypeptide, then this polyclonal antibody can come purifying by immunoaffinity chromatography.The technology that is used to prepare and processes polyclonal antiserum is known in the art.In order to prepare described antibody, the disclosure also provides haptens to change into other amino acid sequence with amino acid sequence or its fragment as animal or human's immunogenic Gpr100.

Monoclonal antibody available from Gpr100 polypeptide or peptide epitopes also can be prepared easily by those skilled in the art.The common method for preparing monoclonal antibody by hybridoma is well-known.The immortal cell line that produces antibody can be set up by Fusion of Cells, also by additive method, for example transforms bone-marrow-derived lymphocyte with carcinogenic dna direct, or sets up with the Epstein-Barr virus transfection.Can screen various character at the monoclonal anti series of orbit epi-position preparation; Be isotype and epi-position affinity.

Monoclonal antibody can use any technology that can prepare antibody molecule by the continuous cell line in the culture to prepare.These include but not limited to, at first hybridoma technology, trisome knurl (trioma) technology, human B cell hybridoma technology (Kosbor et al (1983) the Immunol Today 4:72 that is described by Koehler and Milstein (1975 Nature256:495-497); Cote et al (1983) ProcNatl Acad Sci 80:2026-2030) and EBV-hybridoma technology (Cole et al., MonoclonalAntibodies and Cancer Therapy, pp.77-96, Alan R.Liss, Inc., 1985).

In addition, can use technology, as mouse antibodies gene and human immunoglobulin gene are spliced technology (Morrison et al (1984) the Proc Natl Acad Sci 81:6851-6855 that has the molecule of suitable antigentic specificity and biologic activity with acquisition into preparation " chimeric antibody " development; Neuberger et al (1984) Nature 312:604-608; Takeda et al (1985) Nature 314:452-454).Optional, for the described technology of preparation single-chain antibody (U.S. Patent No. 4,946,779) can be suitable for preparing the specific single-chain antibody of material.

At can being useful especially for diagnosis available from the antibody (monoclonal and polyclonal) of the epi-position of Gpr100 polypeptide or peptide, and neutralizing antibody be useful to passive immunization therapy.Monoclonal antibody especially can be used for exciting anti-idiotype.Anti-idiotype is an immunoglobulin (Ig), and it carries " internal image " of material and/or reagent, need produce protective reaction at described material and/or reagent.It is known in the art being used to excite the technology of anti-idiotype.These anti-idiotypes also can be used for treatment.

Antibody also can be by producing in the inductor in lymphocyte populations or the group of binding reagents by screening recombination immunoglobulin library or high special prepares, as Orlandi et al (1989, ProcNatl Acad Sci 86:3833-3837) and Winter G and Milstein C (1991; Nature 349:293-299) described.

The antibody fragment that also can prepare the specific bond position that comprises polypeptide or peptide.For example, this fragment includes but not limited to, the F that can prepare by the pepsin digested antibody molecule (ab ') ₂Fragment and can be by reduction F (ab ') ₂The disulfide bond of fragment and the Fab fragment for preparing.Perhaps, can make up the Fab expression library and allow fast and easily identify to have required specific monoclonal Fab fragment (Huse WD et al (1989) Science 256:1275-1281).

The technology that is used for manufacture order chain antibody (U.S. Patent No. 4,946,778) also can be transformed to prepare the single-chain antibody of Gpr100 polypeptide.Genetically modified mouse or other biological body comprise that other mammals also can be used to express humanized antibody.

Above-mentioned antibody can be used to separate or identifies the clone that expresses this polypeptide or by this polypeptide of affinity chromatography purification.

The antibody of Gpr100 GPCR polypeptide also can be used to treat the Gpr100 relevant disease.

Diagnostic analysis

Content of the present disclosure also relate to utilize Gpr100 GPCR polynucleotide and polypeptide (with and homologue, variant and derivant) be used for diagnosis or be used for genetic analysis as diagnosticum.With Gpr100 GPCR nucleic acid (comprising homologue, variant and derivant) complementary or can with the nucleic acid of its hybridization, and the antibody of Gpr100 polypeptide also can be used for described analysis.

The detection of the Gpr100 gpcr gene mutant form relevant with dysfunction can add or limit expressing, cross the diagnostic tool of expressing or expressing caused disease of change or disease susceptibility diagnosis because Gpr100 GPCR hangs down providing.The individuality that carries Gpr100 gpcr gene (comprising regulating and controlling sequence) sudden change can be detected at dna level by multiple technologies.

For example, can be from the dna polymorphism pattern of patient's DNA isolation and definite Gpr100.With the pattern identified with known suffer from the mistake of Gpr100-, low-or the patient of the relevant disease of unconventionality expression contrast and make comparisons.Can identify the patient who expresses the genetic polymorphism sexual norm relevant then with the Gpr100 relevant disease.The genetic analysis of Gpr100 gpcr gene can be undertaken by any technology known in the art.For example, can measure the allelic dna sequence dna of Gpr100 and screen individuality by RFLP or snp analysis etc.The existence of dna polymorphism in gene order that can be by detecting Gpr100 or any sequence of regulating and control its expression, identify the patient have with the Gpr100 mistake-, low-or the genetic predisposition of the relevant disease of unconventionality expression.

Therefore can treat the generation of the patient of evaluation like this with prevention Gpr100 relevant disease, or in the early stage further generation or the development that stops this disease more energetically of Gpr100 relevant disease.

The present invention further discloses the kit of the genetic polymorphism sexual norm that is used to identify that the patient is relevant with the Gpr100 relevant disease.This kit comprises the DNA sampling means and measures the means of genetic polymorphism sexual norm, then it is compared with control sample to measure the neurological susceptibility of patient to the Gpr100 relevant disease.Also be provided for diagnosing the Gpr100 relevant disease, comprise the Gpr100 polypeptide and/or at the kit of the antibody of described polypeptide (or its fragment).

Diagnostic nucleic acid can for example obtain from blood, urine, saliva, biopsy or postmortem material from experimenter's cell.In preferred embodiments, this DNA obtains from the haemocyte of the blood pricking patient's finger and collect with thieving paper.In a further preferred embodiment, blood will be at AmpliCard. ^TM. (University of Sheffield, Department of Medicine andPharmacology, Royal Hallamshire Hospital, Sheffield, England S10 2JF) goes up and collects.

DNA can be directly used in detection or can utilize PCR or other amplification techniques to come enzymatic amplification before analysis.The oligonucleotide DNA primer that can prepare special polymorphic dna zone in the target gene of interest is so that realize the amplification of target sequence in the PCR reaction.RNA or cDNA also can be used as template in a similar manner.The dna sequence dna of self-template DNA of increasing then can utilize restriction enzyme analysis, is present in genetic polymorphism in the extension increasing sequence with mensuration, and this patient's genetic polymorphism sexual norm is provided thus.Limited fragment length can be identified by gel analysis.Optional or associating, the technology that can use SNP (single nucleotide polymorphism) for example to analyze.

Disappearance and insertion can detect by the size variation of amplified production with respect to normal genotype.Point mutation can be by identifying the DNA of amplification and the Gpr100 GPCR nucleotide sequence hybridization of mark.Pi Pei sequence can be different from the duplex of mispairing by RNase digestion or melting temperature difference fully.Dna sequence dna difference also can change by the electrophoretic mobility of dna fragmentation in gel, be with or without denaturant or detect by direct dna sequencing.Referring to for example, Myers et al, Science (1985) 230:1242.Sequence variation at ad-hoc location also can be measured by the nuclease protection, and for example RNAse and S1 protection or chemical cleavage method show.Referring to Cotton et al., Proc Natl AcadSci USA (1985) 85:4397-4401.In another embodiment, can make up the oligonucleotide probe array that comprises Gpr100GPCR nucleotide sequence or its fragment and for example carry out, effective screening of genetic mutation.The array technique method is known, has extensive applicability, and can be used at molecular genetics, comprises the various problems in gene expression, genetic linkage and the hereditary variation.(referring to for example: M.Chee et al., Science, Vol 274, pp610-613 (1996)).

Single-strand conformation polymorphism (SSCP) can be used for detecting the difference of electrophoretic mobility between mutant and the wild-type nucleic acid, and (Orita et al. (1989) Proc Natl.Acad.Sci USA:86:2766 is also referring to Cotton (1993) Mutat Res 285:125-144; And Hayashi (1992) Genet AnalTech Appl9:73-79).The single stranded DNA fragment of sample and contrast Gpr100 nucleic acid can and make its renaturation by sex change.The secondary structure of single-chain nucleic acid changes according to sequence, and resulting electrophoretic mobility changes and can detect even the variation of single base.Dna fragmentation can be labeled or detect with label probe.The sensitivity of analyzing can improve by using RNA (rather than DNA), and wherein secondary structure is sensitiveer to the variation of sequence.In preferred embodiments, goal approach uses heteroduple analysis to separate double-stranded heteroduplex molecule (Keen et al. (1991) TrendsGenet 7:5) according to the variation of electrophoretic mobility.

Diagnostic analysis provides the sudden change that detects the Gpr100 gpcr gene by described method to diagnose or measure method to illness such as Gpr100 relevant disease neurological susceptibility.

The existence of Gpr100 GPCR polypeptide and nucleic acid can detect in sample.Therefore, infection of as above listing and disease can be diagnosed by the following method, and this method is included in unusual decline or the rising of determining Gpr100 GPCR polypeptide or Gpr100 GPCR mRNA level in the sample that is derived from the experimenter.This sample can comprise the cell or tissue sample, and it is from suffering from or doubtfully suffering from Gpr100 GPCR that improve, that reduce or that other are unusual the biosome of expressing (comprise expression or pattern in the space or temporal change) relevant disease.As the method that diagnoses the illness, suffer from or the doubtful biosome that suffers from described disease in Gpr100 expression levels or pattern can be effectively with normal biosome in expression levels or pattern compare.

Therefore usually, we have described the method that the nucleic acid that comprises Gpr100 GPCR nucleic acid in the test sample exists, and this method is by with at least a nucleic acid probe of sample contact to described nucleic acid specificity, and monitor the existence of this nucleic acid in the described sample.For example, but this nucleic acid probe specificity and detects between the two combination in conjunction with Gpr100GPCR nucleic acid or its part, also can detect the existence of this compound self.In addition, described the method that Gpr100 GPCR polypeptide exists that detects, this method can be in conjunction with the antibody of this polypeptide by cell sample is contacted, and monitor the existence of this polypeptide in the described sample.This can be by the existence of the compound that forms between antibody and polypeptide of monitoring, or the combination between monitoring polypeptide and the antibody and realizing expediently.The method that detects combination between two entities is known in the art, and comprises FRET (FRET (fluorescence resonance energy transfer)), surface plasmon resonance etc.

Reduction of expressing or raising can utilize any method that is used for quantitative polynucleotide known in this field, measure at rna level as PCR, RT-PCR, RNAse protection, Northern trace and other hybridizing methods.Can be used for being determined at the protein in the sample that is derived from the host, is well known to a person skilled in the art as the analytical technology of Gpr100 GPCR level.Described determination method comprises that radiommunoassay, competitive binding assay, Western engram analysis and ELISA measure.

This paper relates to the diagnostic kit that is used for disease or disease susceptibility (comprising infection), and described disease for example obesity, appetite inhibition, metabolism disorder, appetite suppresses.This diagnostic kit comprises Gpr100GPCR polynucleotide or its fragment; Complementary nucleotide sequence; The antibody of Gpr100 GPCR polypeptide or its fragment or Gpr100 GPCR polypeptide.

Chromosome analysis

Nucleotide sequence described herein is also identified very valuable to chromosome.Ad-hoc location on this sequence specific ground target wall scroll human chromosome and can with its hybridization.As mentioned above, people Gpr100 GPCR finds to be positioned human chromosome 1q22.

It is the important first step that those sequences are associated with disease related gene that correlated series is positioned on the chromosome.In case sequence has been positioned accurate chromosome position, the physical location of this sequence on chromosome just has been associated with the genetic map data.Described data are searched among the Mendelian heritance in Man (by the online acquisition of Johns Hopkins University Welch MedicalLibrary) for example at V.McKusick.Determine to be positioned the gene of identical chromosomal region and the relation between the disease by linkage analysis (the common heredity of the adjacent gene of physics) subsequently.

Also can measure infection and the individuality that do not infect between the difference of cDNA or genome sequence.If at the individuality of some or all of infection, rather than observe sudden change in any normal individuality, then this sudden change may be the pathogenic agent of disease.

Prevention and methods of treatment

This paper provides the method for the treatment relevant unusual state of an illness excessive with Gpr100 GPCR activity and in shortage.

If Gpr100 GPCR's is active excessive, can utilize several method.A kind of method comprises to accepting carrier on the aforesaid inhibitor compound (antagonist) of experimenter's effective dose and the materia medica with combining by block ligand and Gpr100 GPCR, or, alleviate unusual condition thus and suppress activation by suppressing secondary signal.

In the another kind method, can use the Gpr100 GPCR polypeptide that still can compete the soluble form of binding partner with endogenous Gpr100 GPCR.The typical embodiments of described competitor comprises the fragment of Gpr100GPCR polypeptide.

Also have in a kind of method, the expression of gene of this endogenous Gpr100 GPCR that encodes can suppress with the expression interrupter technique.Known described technology comprises in the mode of inside generation or separate administration uses antisense sequences.Referring to for example, O ' Connor, JNeurochem (1991) 56:560 inOligodeoxvnucleotides as Antisense 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., Nucleic Acids Res (1979) 6:3073; Cooney et al., Science (1988) 241:456; Dervan et al., Science (1991) 251:1360.But these oligomer self are used or the oligomer of being correlated with can be expressed in vivo.

To express and the active relevant unusual state of an illness in order treating, also can to use several method with Gpr100 GPCR is low.A kind of method comprise to experimenter's administering therapeutic effective dose with materia medica on can accept the compound of the activation Gpr100 GPCR of carrier coupling, promptly aforesaid activator alleviates this unusual condition thus.Perhaps, gene therapy can be used for influencing the endogenous production of Gpr100 GPCR by the relevant cell among the experimenter.As discussed above, for example can transform the Gpr100 polynucleotide and in the replication defect type retroviral vector, express.The separable subsequently incasing cells that changes the retrovirus expression construct and import the retroviral plasmid vector transfection of using the RNA that comprises coding Gpr100 polypeptide makes this incasing cells now can produce the infectious viral particle that comprises interested gene.These can be produced cell is applied to the experimenter and is used in the body engineered cells and express polypeptide in vivo.The summary of gene therapy is referring to Chapter 20, Gene Therapy and other Molecular Genetic-basedTherapeutic Approaches, (with the list of references of wherein quoting) in Human MolecularGenetics, T Strachan and A P Read, BIOS Scientific Publishers Ltd (1996).

Preparation and administration

Peptide, for example the Gpr100 GPCR polypeptide of soluble form and activator and antagonist peptide or micromolecule, can with the suitable pharmaceutical carrier co-formulated.Described preparation comprises polypeptide or compound and the pharmaceutical acceptable carrier or the excipient for the treatment of effective dose.Described carrier includes but not limited to, salt solution, buffer saline, glucose, water, glycerine, ethanol and combination thereof.Preparation should be fit to the mode of administration, and is well known by persons skilled in the art.We have further described the drug packages and the kit of one or more container that comprises one or more composition that is full of above-mentioned composition.

Polypeptide and other compounds can use separately or with other compounds, as treatment compound coupling.

The preferred form of whole body administration medicine composition comprises injection, usually by intravenous injection.Also can use other injecting pathways, in for example subcutaneous, intramuscular or the peritonaeum.The alternative that is used for systemic administration comprises utilizes bleeding agent, strides mucous membrane and applied dermally as cholate or fusidic acid or other detergents.In addition, if suitably be formulated as the preparation of enteric or capsulation, oral also passable.These compounds also can and/or localize (localize) through local (topical), with ointment (salve), and paste (paste), forms such as gel are used.

The required dosage scope depends on selected peptide, route of administration, preparation nature, and the character of experimenter's state of an illness and attending doctor's judgement.Appropriate dosage is 0.1-100 μ g/kg experimenter.Consider the different efficient of obtainable classes of compounds and different administration approach, the expection required dosage can have bigger variation.For example expect that the dosage that oral administration needs is higher than intravenous injection.As known in the art, can utilize the conventional difference of regulating these dosage levels of the standard experience that is used to optimize.

The often form of therapy of aforesaid to be called " gene therapy " and endogenous generation in the experimenter of the polypeptide that is used for the treatment of.Therefore for example, can use polynucleotide from experimenter's cell, for example DNA or RNA transform, and for example utilize retroviral plasmid vector and the interior coded polypeptide of earlier external back body.This cell imports among the experimenter subsequently.

Pharmaceutical composition

This paper also provides pharmaceutical composition, comprises Gpr100 polypeptide, polynucleotide, peptide, carrier or antibody and optional pharmaceutical acceptable carrier, thinning agent or the excipient (comprising its combination) for the treatment of effective dose.

Pharmaceutical composition can be used for the human or animal in physianthropy and veterinary science, it comprises any one or more pharmacy acceptable diluent, carrier or excipient usually.The carrier accepted or the thinning agent that are used for the treatment of purposes are that pharmaceutical field is known, at for example Remington ' s PharmaceuticalSciences, describe among the Mack Publishing Co. (A.R.Gennaro edit.1985).The selection of pharmaceutical carrier, excipient or thinning agent can be considered that the approach that is intended to use and standard pharmaceutical are put into practice and select.This pharmaceutical composition can comprise as carrier, excipient or thinning agent or any suitable bonding except that carrier, excipient or thinning agent, lubricant, suspending agent, coating agent (coating agent), solubilizer.

Antiseptic, stabilizing agent, dyestuff and 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.

Has different compositions/preparation demand according to different delivery systems.For instance, this pharmaceutical composition can be formulated as and utilize micropump (mini-pump) or deliver by mucosal route, for example as nasal spray or inhalation aerosol or deglutible solution, or parenteral is delivered, wherein said composition is formulated as injectable form, delivers by for example intravenous, intramuscular or subcutaneous approach.Perhaps, said preparation can be designed to deliver by these two kinds of approach.

When this reagent by gastrointestinal mucosa when mucous membrane is delivered, it should be able to be by keeping stable during the intestines and stomach; For example, it should be able to resist proteoclastic degradation, and is stable at acid pH, and the detergent effect of opposing bile.

In the time of suitably, this pharmaceutical composition can be used in the following way: through sucking, form with suppository or pessary (pessary), with washing lotion, solution, emulsifiable paste, the form local application of ointment or fine powder (dusting powder), by utilizing lagging agent (skin patch), oral with the tablet form that contains excipient such as starch or lactose, or in capsule or avette capsule (ovule) separately or with excipient, or with contain seasoning or colorant elixir, the form of solution or suspension, or outside stomach and intestine such as, for example through intravenous, in the muscle or hypodermic injection.For parenteral, said composition is preferably used in the form of aseptic aqueous solution, and it can comprise other material, and for example enough salt or monose are so that this solution and blood etc. ooze.For the administration in cheek or hypogloeeis, said composition can be used with the tablet of usual manner preparation or the form of lozenge.

Vaccine

Another embodiment relates to the method for induction of immunity reaction in mammal; it comprises with enough Gpr100 GPCR polypeptide or its fragment seeded with mammalian of generation antibody and/or T cellullar immunologic response, avoids obesity, appetite inhibition, metabolism disorder and other to protect described animal.

Another embodiment relates to the method for induce immune response in mammal; it comprises by means of the carrier that instructs Gpr100 GPCR polynucleotide expression in vivo delivers Gpr100 GPCR polypeptide, thereby induce immune response is to produce the antibody that the described animal of protection avoids disease.

Further embodiment relates to immunity/bacterin preparation (composition), it is when importing mammalian hosts, induce the immune response at Gpr100 GPCR polypeptide in this mammal, wherein said composition comprises Gpr100 GPCR polypeptide or Gpr100 gpcr gene.This bacterin preparation can further comprise suitable carriers.

Because Gpr100 GPCR polypeptide can decompose under one's belt, it is preferably used by parenteral (comprising injections such as subcutaneous, intramuscular, intravenous, intracutaneous).The preparation that is suitable for parenteral administration comprises water-based and anhydrous aseptic parenteral solution, and it can comprise anti--oxygenant, damping fluid, bacteriostatic and make the solute that said preparation and recipient's blood etc. oozes; And the water-based and the anhydrous sterile suspensions that can comprise suspending agent or thickening agent.Said preparation can be present in unit dose or the multi-dose container, for example Mi Feng ampoule and bottle, and can be kept under the condition of freeze-drying, only need add immediately before use aseptic liquid-carrier.This bacterin preparation also can comprise and be used to strengthen the immunogenic adjuvant system of said preparation, for example oil-in-water system and other system known in the art.Dosage will depend on the specific activity of vaccine, and can measure easily by normal experiment.

Vaccine can be from one or more Gpr100 polypeptide or peptide preparation.

Comprising immunogenic polypeptide or peptide is well known by persons skilled in the art as the preparation of the vaccine of active component.Usually, described vaccine production is injectable liquid solution or suspending liquid; Also can prepare the solid form that is suitable for before injection, being dissolved or suspended in the liquid.Said preparation also can be emulsified, and perhaps protein is wrapped in the liposome.Active immunogenic components often and pharmacy is acceptable and the mixed with excipients compatible with this active component.Suitable excipient is for for example, water, salt solution, glucose, glycerine, ethanol etc. and combination thereof.

In addition, if desired, this vaccine can comprise more a spot of auxiliary substance, as the adjuvant of wetting agent or emulsifying agent, pH buffering agent and/or enhancing vaccine validity.Effectively the example of adjuvant includes but not limited to: aluminium hydroxide; N-acetyl group-muramyl-L-Threonyl-D-isoglutamine (thr-MDP); N-acetyl-demethyl-muramyl-L-alanyl-D-isoglutamine (CGP11637; be called demethyl-MDP); the different glutamyl of N-acetyl group muramyl-L-alanyl-D--L-alanine-2-(1 '-2 '-two palmityls-sn-glyceryl-3-hydroxyl phosphorus acyloxy)-ethamine (CGP 19835A; be called MTP-PE); and RIBI; it comprises the three kind compositions of extraction from bacterium, monophosphoryl lipid A in 2% squalene/Tween 80 emulsions; trehalose dimycolate and cell membrane bone (MPL+TDM+CWS).

Adjuvant and the other example of other reagent comprise aluminium hydroxide, aluminum phosphate, aluminium potassium sulfate (alum), beryllium sulfate, silica, porcelain earth, carbon, water-in-oil emulsion, oil-water emulsifiers, muramyl dipeptide, bacterial endotoxin, lipid X, CBP (propionibacterium acne (Propionobacteriumacnes)), Bordetella pertussis, polyribonucleotide, sodium alginate, sheep oil, lysolecithin, vitamin A, saponin(e, liposome, levamisol, the DEAE-dextran, segmented copolymer or other synthetic adjuvant.Described adjuvant can be from various sources commercial the acquisition, for example, Merck Adjuvant65 (Merck and Company, Inc., Rahway, N.J.) or incomplete Freund and Freund's complete adjuvant (Difco Laboratories, Detroit, Michigan).

Usually, use the adjuvant of the potpourri of for example Amphigen (oil-in-water), Alhydrogel (aluminium hydroxide) or Amphigen and Alhydrogel.Have only the aluminium hydroxide approval to be used for the mankind.

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

After the preparation, this vaccine can mix in the sterile chamber of sealing subsequently, and in low temperature, for example 4 ℃ of preservations, but or freeze-drying.Desivac is allowed with stable form standing storage.

This vaccine parenteral as usual gives, by injection, for example subcutaneous or intramuscular injection.The additional formulations that is suitable for other administering modes comprises suppository, and is oral formulations in some cases.For suppository, can comprise conventional bonding agent and carrier, for example, poly alkylene glycol or triglyceride; Described suppository can be from containing 0.5% to 10%, and the potpourri of the active component of preferred 1% to 2% scope forms.Oral formulations comprises normally used excipient, for example the sweet mellow wine of pharmaceutical grade, lactose, starch, dolomol, saccharin sodium, cellulose, magnesium carbonate etc.These compositions are taked the form of solution, suspending liquid, tablet, pill, capsule, sustained release formulation or pulvis, and comprise 10% to 95%, preferred 25% to 70% active component.When vaccine combination is cryodesiccated, this cryodesiccated material can reconstruct before administration, for example is suspending liquid.Reconstruct preferably realizes in damping fluid.

Being used for the oral capsule of patient, tablet and pill can have enteric coating, comprises for example Eudragit " S ", Eudragit " L ", acetyl cellulose, Cellulose Acetate Phthalate ester or HPMC ester.

The Gpr100 polypeptide can be prepared the vaccine that becomes neutrality or salt form.The acceptable salt of pharmacy comprises for example hydrochloric acid or phosphoric acid or the organic acid salt of acetate, oxalic acid, tartrate and maleic acid formation for example of the salt (forming with the free amine group of peptide) of sour addition and itself and mineral acid.The salt that forms with free carboxy also can derive from inorganic base, for example NaOH, potassium hydroxide, ammonium hydroxide, calcium hydroxide or ferric hydroxide, and organic base.For example isopropylamine, trimethylamine, 2-ethylamino-ethanol, histidine and procaine.

Use

Usually, the doctor can determine to be suitable for most the actual dose of individual subjects, and it will change along with concrete patient's age, body weight and reaction.Following dosage is the example of average case.Certainly having wherein higher or lower dosage range all is useful individual cases.

This medicine and vaccine combination can be used by direct injection.Said composition can be prepared and be used for parenteral, mucous membrane, intramuscular, intravenous, subcutaneous, intraocular or using through skin.Usually, every kind of protein can 0.01 to 30mg/kg body weight, and preferred 0.1 to 10mg/kg, and more preferably the dosage of 0.1 to 1mg/kg body weight is used.

Term administering " comprise by the technology of virus or non-virus and delivering.The delivery mechanism of virus includes but not limited to adenovirus vector, adeno-associated virus (AAV) carrier, herpesvirus vector, retroviral vector, slow virus carrier and baculovirus vector.The delivery mechanism of non-virus comprises transfection, liposome, immunoliposome, the lipofection reagent (lipofectin) of lipid mediation, the amphipathic molecule (CFAs) and the combination thereof of cationic surface.The described used approach of mechanism of delivering includes but not limited to approach that is mucous membrane, nose, the oral cavity, parenteral, stomach and intestine, local or the hypogloeeis.

Term administering " approach by mucous membrane that includes but not limited to delivers, for example as nasal spray or gasoloid so that suck or as deglutible solution; Parenteral approach is wherein delivered by injectable form (for example intravenous, intramuscular or subcutaneous approach).

Term " is used jointly " and for example is meant every kind of Gpr100 polypeptide and additional entities, makes it possible to achieve immune necessary the adjusting as the medicine-feeding part and time of adjuvant.Therefore, although polypeptide can be used in the identical time in time and in same area with adjuvant, it may be favourable using polypeptide with time different with adjuvant and different positions.This polypeptide is with adjuvant even can deliver in identical delivery carrier, but and this polypeptide and antigen coupling and/or not coupling and/or coupling and/or not coupling in heredity.

Described polypeptide, polynucleotide, peptide, nucleotide, described antibody and optional adjuvant can single doses or use or be applied to jointly host experimenter respectively with multiple dose.

Vaccine combination can be used by many different approach with pharmaceutical composition, in for example injection (it comprises parenteral, the subcutaneous and intramuscular injection) nose, the giving of mucous membrane, the oral cavity, intravaginal, urethra or eye.

This vaccine and pharmaceutical composition be parenteral administration as usual, by injection, for example subcutaneous or intramuscular injection.The additional formulations that is suitable for other administering modes comprises suppository, and is oral formulations in some cases.For suppository, can comprise conventional bonding agent and carrier, for example, poly alkylene glycol or triglyceride; Described suppository can be from containing 0.5% to 10%, and the potpourri of the active component of preferred 1% to 2% scope forms.Oral formulations comprises normally used excipient, for example the sweet mellow wine of pharmaceutical grade, lactose, starch, dolomol, saccharin sodium, cellulose, magnesium carbonate etc.These compositions are taked the form of solution, suspending liquid, tablet, pill, capsule, sustained release formulation or pulvis, and comprise 10% to 95%, preferred 25% to 70% active component.When vaccine combination is cryodesiccated, this cryodesiccated material can be reconstructed into before administration, for example suspending liquid.Reconstruct preferably realizes in damping fluid.

Perhaps, treatment compound of identifying by method described herein or the reagent treatment or the prevention that can be used for diabetes associated conditions or body weight associated conditions.In one aspect, compound or reagent can be natural, synthetic, semisynthetic or Gpr100 acceptor gene, Gpr100 acceptor gene product or its fragment of reorganization and the analog of this gene, gene outcome or fragment.Another aspect, compound can be specific antibody, antisense DNA or RNA or the organic or inorganic micromolecule of gene or gene outcome.In an embodiment preferred, compound or reagent are influential to activity, expression or the function of Gpr100 acceptor gene or Gpr100 acceptor gene product.

Be provided for the methods of treatment of diabetes associated conditions or body weight associated conditions.In one aspect, give the experimenter who needs it with the agent administration that can regulate the Gpr100 acceptor of treatment effective dose.The reagent that can regulate the Gpr100 acceptor includes but not limited to specific antibody, antisense DNA or RNA or the organic or inorganic micromolecule of gene or gene outcome.The Gpr100 receptor modulators can be independent, or use as the part that pharmacy can be accepted composition.For example, the Gpr100 receptor modulators can with other Gpr100 receptor stimulating agents or antagonist or co-administered with other pharmaceutically active substances.For example, other pharmaceutically active substances can comprise known in the art resisting-diabetes reagent or anti--obesity reagent, or is used for the reagent of other symptoms or disease treatment.

The method that is used for the treatment of diabetes associated conditions or body weight associated conditions comprises that Gpr100 acceptor gene or Gpr100 acceptor with the treatment effective dose are administered to the experimenter who needs it.

Many-sided

State in many-sided and the paragraph that embodiment is numbered hereinafter now of the present invention; Can understand and the present invention includes these aspects:

Paragraph 1. comprises the Gpr100 GPCR polypeptide of amino acid sequence shown in SEQ ID NO.3 or the SEQ ID NO:5 or its homologue, variant or derivant.

The nucleic acid of the polypeptide of paragraph 2. coding paragraphs 1.

The nucleic acid of paragraph 3. paragraphs 2 comprises nucleotide sequence or its homologue, variant or derivant shown in SEQ ID NO.1, SEQ ID No.2 or the SEQ ID NO:4.

Paragraph 4. comprises the polypeptide of the fragment of paragraph 1 polypeptide.

The polypeptide of paragraph 5. paragraphs 3, it is included in one or more zones of homology between SEQ ID No.3 and the SEQ ID No.5, or it is included in one or more zones of allos between SEQ ID No.3 and the SEQ ID No.5.

The nucleic acid of paragraph 6. coding paragraphs 4 or 5 polypeptide.

Paragraph 7. comprises the carrier of the nucleic acid of paragraph 2,3 or 6.

Paragraph 8. comprises the host cell of the carrier of the nucleic acid of paragraph 2,3 or 6 or paragraph 7.

Paragraph 9. comprises the transgenic nonhuman animal of the carrier of the nucleic acid of paragraph 2,3 or 6 or paragraph 7.

The transgenic nonhuman animal of paragraph 10. paragraphs 9, it is a mouse.

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

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

Paragraph 13. is identified the method for Gpr100 GPCR antagonist, and this method comprises and will express the cells contacting candidate compound of Gpr100 acceptor, and measures in the cell and whether encircle the level of AMP (cAMP) owing to described contact reduces.

Paragraph 14. is identified the method that can reduce the compound of ring AMP endogenous levels in the cell, and this method comprises and will express the cells contacting candidate compound of Gpr100 GPCR, and measures in the cell and whether encircle the level of AMP (cAMP) owing to described contact reduces.

Paragraph 15. is identified can be in conjunction with the method for the compound of Gpr100 GPCR polypeptide, and this method comprises whether Gpr100 GPCR polypeptide is contacted and measure this candidate compound in conjunction with Gpr100 GPCR polypeptide with candidate compound.

Paragraph 16. is by the arbitrary method compounds identified of paragraph 11 to 15.

Paragraph 17. energy specificitys are in conjunction with the compound of the polypeptide of paragraph 1,4 or 5.

Paragraph 18. paragraphs 1,4 or 5 polypeptide or the purposes of nucleic acid in preparing the method for antibody of its part or paragraph 2,3 or 6.

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

Paragraph 20. pharmaceutical compositions comprise following any or multiple and pharmaceutical acceptable carrier or thinning agent: paragraph 1,4 or 5 polypeptide or its part; Paragraph 2,3 or 6 nucleic acid or its part; The carrier of paragraph 7; The cell of paragraph 8; Paragraph 16 or 17 compound; And the antibody of paragraph 19.

Paragraph 21. vaccine combinations comprise following any or multiple: paragraph 1,4 or 5 polypeptide or its part; Paragraph 2,3 or 6 nucleic acid or its part; The carrier of paragraph 7; The cell of paragraph 8; Paragraph 16 or 17 compound; And the antibody of paragraph 19.

Paragraph 22. is used for the diagnostic kit of disease or disease susceptibility, comprises following any or multiple: paragraph 1,4 or 5 polypeptide or its part; Paragraph 2,3 or 6 nucleic acid or its part; The carrier of paragraph 7; The cell of paragraph 8; Paragraph 16 or 17 compound; And the antibody of paragraph 19.

Paragraph 23. treatment suffers from the method with the patient of the active relevant disease of the Gpr100 GPCR that strengthens, and this method comprises and gives patient Gpr100 the antagonist of GPCR.

Paragraph 24. treatment suffers from the method with the patient of the active relevant disease of the Gpr100 GPCR that reduces, and this method comprises and gives patient Gpr100 the activator of GPCR.

Paragraph 25. paragraphs 23 or 24 method, wherein Gpr100 GPCR comprises having polypeptide of sequence shown in SEQ IDNO:3 or the SEQ ID NO:5.

Paragraph 26. is used for the treatment of and/or prevents the method for disease among the patient, and it comprises the step with following any or multiple patient of giving: paragraph 1,4 or 5 polypeptide or its part; Paragraph 2,3 or 6 nucleic acid or its part; The carrier of paragraph 7; The cell of paragraph 8; Paragraph 16 or 17 compound; The antibody of paragraph 19; The pharmaceutical composition of paragraph 20; And the vaccine of paragraph 20.

Paragraph 27. reagent comprise polypeptide or its part of paragraph 1,4 or 5; Paragraph 2,3 or 6 nucleic acid or its part; The carrier of paragraph 7; The cell of paragraph 8; Paragraph 16 or 17 compound; And/or the antibody of paragraph 19, described reagent be used for the treatment of or prophylactic method in.

Paragraph 28. paragraphs 1,4 or 5 polypeptide or its part; Paragraph 2,3 or 6 nucleic acid or its part; The carrier of paragraph 7; The cell of paragraph 8; Paragraph 16 or 17 compound; And the antibody of paragraph 19 is used to prepare the purposes of treatment or prophylactic pharmaceutical composition.

Paragraph 29. non-human transgenic animals are characterised in that these transgenic animals comprise the Gpr100 gene of change.

The inhuman transgenic animals of paragraph 30. paragraphs 29, wherein said variation be selected from the group of following composition: Gpr100 disappearance, cause the Gpr100 sudden change of afunction, the foreign gene that will have a nucleotide sequence of target or random mutation to import Gpr100, to import Gpr100 from the foreign gene of another species, and the combination of any of these.

Paragraph 31. has the non-human transgenic animal of the destroyed endogenous Gpr100 gene of function, and wherein these transgenic animals comprise and express the transgenosis of coding allos Gpr100 albumen in its genome.

Paragraph 32. is used for the nucleic acid construct of the Gpr100 gene of functional destruction host cell, and this nucleic acid construct comprises: (a) nonhomologous replacing section; (b) be positioned at first homologous region of non-homogeneous replacing section upstream, this first homologous region has and the essentially identical nucleotide sequence of a Gpr100 gene order, and second homologous region that (c) is positioned at non-homogeneous replacing section downstream, this second homologous region has and the same substantially nucleotide sequence of the 2nd Gpr100 gene order, and described the 2nd Gpr100 gene order is positioned at the downstream of first Gpr100 gene order in naturally occurring endogenous Gpr100 gene.

Paragraph 33. is used to produce the method for Gpr100 GPCR polypeptide, and this method is included in the host cell of cultivating paragraph 8 under the condition of the nucleic acid of expressing coding Gpr100 GPCR polypeptide.

The method that paragraph 2,3 or 6 nucleic acid exist in paragraph 34. test sample, this method comprise at least a nucleic acid probe of sample contact to described nucleic acid specificity, and monitor the existence of this nucleic acid in the described sample.

The method that paragraph 1,4 or 5 polypeptide exist in paragraph 35. test sample, this method comprises the antibody that sample is contacted paragraph 19, and monitors the existence of this polypeptide in the described sample.

Paragraph 36. diagnosis are expressed by Gpr100 GPCR and are improved, reduce or cause unusually in addition, or relative disease or syndromic method, this method comprises the steps: that (a) detects and to suffer from or doubtfully suffer from obesity, comprise the prevention of obesity or weight increase, appetite suppresses, the lipid metabolism illness, comprise hyperlipidemia, dyslipidemia and hypertriglyceridemia, diabetes and associated conditions include but not limited to: type ii diabetes, impaired glucose tolerance, insulin resistance syndrome, the X syndrome, hyperglycemia, acute pancreatitis, angiocardiopathy, hypertension, the expression of Gpr100 GPCR or pattern in the animal of cardiomegaly and hypercholesterolemia; (b) making comparisons with this expression or pattern and intact animal.

Embodiment

Embodiment 1. transgenosis Gpr100 knock-out mices

The structure of Gpr100 gene targeting vector

Utilize the homology search of genome database to identify the Gpr100 gene with the biological information method.The genome contig of assembling 62kb from the plurality of data storehouse.This contig provides enough flanking sequence information so that can design the homology arm that the clone enters targeting vector.

The Gpr100 gene of mouse has 1 coding extron.Design target strategy comprises most of membrane spaning domains to remove most of coded sequence., and fragment cloning gone in the targeting vector at 5 ' homology arm of the 3.1kb that waits to lack regional both sides and 3 ' homology arm of 1.8kb by pcr amplification.5 ' end of each Oligonucleolide primers of the synthetic described arm that is used to increase, different cleavage sites with (rare-cuting) restriction enzyme of comprising the cleavage site rareness, the cloning site fit of described cleavage site and carrier polylinker, but in described arm, do not exist.With regard to Gpr100, design as the listed primer of following primer table, described primer has 5 ' arm cloning site of NotI/SpeI and 3 ' arm cloning site of AscI/FseI (structure of employed targeting vector comprises relevant restriction site, is presented among Fig. 2).

Except the primer of arm to (5 ' armF/5 ' armR) and (3 ' armF/3 ' armR), for the following purpose design more primers special to the Gpr100 locus: 5 ' and 3 ' probe primer to (5 ' prF/5 ' prR and 3 ' prF/3 ' are prR), be used for increasing outside each arm and extend beyond the 150-300bp fragment of two weak points of the non-duplicate factor group DNA of each arm, thereby allow the Southern that in the target of the inferring clone who separates, carries out this target gene seat to analyze; The primer of mouse Genotyping is to (hetF and hetR), and described primer can be distinguished wild type, heterozygosis and homozygous mouse when using in the multiplex PCR that utilizes carrier specificity primer (being Asc350 in this case); And be that target screens primer (3 ' scr) at last, it is in the annealing of downstream of 3 ' arm region end, and when its specific 1.9kb amplified matter of (being Asc53 in this case) generation target incident (target event) when the special primer of carrier (TK5IBLMNL) 3 ' end is matched.This amplified matter can only be derived from and the template DNA that required genome changes, also allows to identify the cell of correct target from clone's background that the random integration that comprises carrier copies occur.SEQ ID NO:19 shows the position of these primers that are used for the target strategy and the genome structure in Gpr100 locus zone.

Table 1.Gpr100 primer sequence

TTGTGCAGAGTTCAATGGAGAATGTTG-SEQ ID

musGpr100C 5′prF No.6

CCAGAAACACTCTACGCCTGTCACCTG-SEQ ID

musGpr100C 5′prR No.7

musGpr100C TttgcggccgcAAAGTGACTCATGCTGCTCCCATCTTC-

5′armF Not SEQ ID No.8

musGpr100C AaaactagTCCCAGCAAGCCAATGATACCTACAAG -

5′armR Spe SEQ ID No.9

musGpr100C TttggcgcgCCTGGGACAGTACTTTCTACACCTTTC -

3′armF Asc SEQ ID No.10

musGpr100C TttggccggccTCCATTTAAGAAGAGATCTTGAGCCAG

3′armR Fse -SEQ ID No.11

TGGATCCTTTTATTTTGGAGACTGAAC-SEQ ID

musGpr100C 3′scr No.12

CCTGGCTCAAGATCTCTTCTTAAATGG-SEQ ID

musGpr100C 3′prF No.13

GGTGAGCAATCAGATCATGAGACTTAC-SEQ ID

musGpr100C 3′prR No.14

GCTTACCAGCTACAGAGGGTAGTTCTG-SEQ ID

musGpr100C hetF No.15

TGATGGGAAGGATGTAAGTATGAAAGGTG-SEQ

musGpr100 hetR3 ID No.16

GTCGTGACCCATGGCGATGCCTGCTTG-SEQ ID

Asc350 No.17

CGGATCCACTAGATAACTTCGTATAGC-SEQ ID

Asc53 No.18

The position of selecting homology arm is with functional destruction Gpr100 gene.The preparation targeting vector, the non-homogeneous sequence replacing that report (being independent of the lacZ gene of frame) is formed is expressed by the endogenous gene of selecting the box upstream in Gpr100 wherein to be lacked zone, this select box by with (promoted) neomycin phosphotransferase (neo) genomic constitution of tape starting of Gpr100 gene equidirectional arrangement.

In case 5 ' and 3 ' homology arm is cloned into targeting vector TK5IBLMNL, just can utilize standard molecular biological technique to prepare a large amount of highly purified DNA goods.With the no endotoxic DNA of the 20 μ g prepared fresh restriction enzyme PmeI cutting with another cleavage site rareness, described site is unique between ampicillin resistance gene and the bacterium origin of replication in carrier framework.Precipitate linearizing DNA subsequently and be resuspended in the 100 μ l phosphate buffered saline (PBS)s, prepare to be used for electroporation.

Behind the electroporation 24 hours, in the nutrient culture media that contains 200 μ g/ml neomycins, cultivated cells transfected 9 days.Before identifying the clone that homologous recombination wherein takes place between endogenous Gpr100 gene and target construct by PCR screening (utilizing primer 3 ' scr and Asc53 as mentioned above), the clone is being chosen into 96 orifice plates, duplicate and increase.Ratio that can 1 to 5% is identified positive colony.Increase these clones so that duplicate is freezing, prepare enough high-quality DNA and prepare the Southern trace, described Southern trace be used to use as mentioned above the outside 5 of preparation ' and 3 ' probe confirm this target incident, these all use standard method (Russ et al, Nature 2000 Mar 2; 404 (6773): 95-99).When during with outside probe hybridization, confirming the ES cell clone of homology target by the existence of sudden change band and unaltered wild type band with the Southern trace of the DNA of diagnostic restriction enzyme digestion.For example, utilize 5 ' probe, the genomic DNA that digests with SpeI can produce the wild type band of 15.7kb and the target band of 8.5kb; And with 3 ' probe, the DNA of SpeI cutting will produce the wild type band of 15.7kb and the target band of 11.5kb.

The generation of Gpr100 GPCR deficient mice

C57BL/6 is female to carry out mating with male mice, at the 3.5th day separation blastocyst of gestation.To 10-12 the cell of each blastocyst injection from selected clone, and with 7-8 the female uterus of blastocyst implantation false pregnancy F1.The a brood of chimeric cub of being born, described cub comprise some high levels (reaching 100%) Slate greys (agouti) male (the Slate grey hair color shows the influence of derivation from target clone's cell).These male chimeras and female MF1 and 129 mouse carry out mating, determine the system genitale transmission by the Slate grey hair color with by the pcr gene somatotype respectively.

The pcr gene somatotype utilizes primer hetF and hetR to carry out with the third carrier specificity primer (Asc350) on the afterbody intermediate plate of cracking.This multiplex PCR allows from wild type gene seat (if existence) thereby from primer hetF and hetR amplification generation 285bp band.The hetF site lacks in knock-out mice, so can't carry out this amplification from target allele.Yet the Asc350 primer will be united with the hetR primer that is positioned at the annealing of 3 ' arm area inside just, from the band of target gene seat amplification 397bp.Therefore, the following genotype that shows cub of this multiplex PCR: the wild type sample shows the band of wall scroll 285bp; The DNA sample of heterozygosis produces two bands at 285bp and 397bp; And homozygous sample is with the specific 397bp band of a display target.

The destroyed transgenic mice of Gpr100 acceptor gene presents metabolic disorder.Particularly, after the contact high fat diet, relative wild type control mice, transgenic mice body weight, height and body fat increase less, and the destruction of prompting Gpr100 acceptor can respond some resistance that high fat diet produces to be increased weight increase or body fat.Can provide the valuable understanding that treats and/or prevents to the resistance of weight increase to associated conditions such as diabetes and obesity.Thereby the Gpr100 acceptor can be used as target and is used to find treat the therapeutic agent of diabetes associated conditions.

Embodiment 2. biological datas: serum chemistry: blood

Collect sample to syringe by the tip cardiac puncture.Every kind of whole blood sample of one hectolambda is transferred in the pipe of filling EDTA in advance.Remaining blood sample is by the centrifugal serum that is transformed in the serum tube that has the gel separation device.Every kind of blood serum sample of analysis as described below subsequently.Collect the tip blood sample of old mouse to kapillary by back eye socket venipuncture.This method produces about 200 μ L whole bloods, and it is transferred to the serum tube that has the gel separation device and is used for serum chemistry analysis (seeing below), or is transferred in the pipe of filling EDTA in advance and is used for analysis of Hematology Changes.

Utilize the laboratory technique of standard and the following parameters of determination method serum analysis: insulin, alanine aminotransferase, albumin, alkaline phosphatase, aspartate transaminase, hydrocarbonate, total bilirubin, blood urea nitrogen (BUN), calcium, chloride, cholesterol, creatine kinase, creatinine, globulin, glucose, high-density lipoprotein (HDL) (HDL), lactic dehydrogenase, low-density lipoprotein (LDL), osmolality, phosphorus, potassium, total protein, sodium and triglyceride.

Embodiment 3. biological datas: histology and density analysis

Adipose tissue histology after the fasting

Mouse (for mutant and wild type, n=4) fasting 16h, kill, dissect the white adipose tissue subsequently, according to the Histological method of standard, be fixed in 4% paraformaldehyde, be embedded in the wax, cut into slices and utilize h and E dyeing.

The result shows in Fig. 4.Fig. 4 shows the white adipose tissue from 4 mutant and 4 wild types, and last figure demonstration contrasts not fasting animal, and 3 figure in bottom show the tissue from the fasting animal.

Data show that the cell of wild-type mice adipocyte size reduces after the fasting.In knock-out mice, do not observe this reduction, therefore show to knock out fat is flowed.

Densitometry

Kill mouse and utilize Piximus ^TMDensitometric analysis.Make mouse be exposed to high-energy and low-energy X-ray beam with x-ray source.High-energy and low-energy attenuation ratio make bone and soft tissue and separate with fat with lean meat in the tissue sample.Obtain and the recording density continuous data, described data comprise that bone mineral density is (with g/cm ²The BMD of expression), bone mineral content (BMC that represents with g), bone and the number percent (representing) of organizing area, total liver mass and fat to account for the health soft tissue with fatty %.

When comparing with the control mice of age and gender matched, homozygous mutant mouse presents the number percent (fatty %) that fat accounts for the health soft tissue and increases.In the female homozygous mutant mouse in about 49 day age, observe increasing of this percent fat.When being given normal diet (non-high fat diet), mouse further sees increasing of percent fat.

Measure: whole high fat diet duration of exciting record height and body weight.

The result: knock-out mice presents the metabolic characteristics of diabetes and obesity.

Carry out high fat diet and excite about 8 weeks knocking out mouse, and carry out the glucose tolerance check.High fat diet excites back also recording density measurement and body weight and height (measurement).

Dextrose tolerance test (GTT): about 5 hours of mouse fasting and the injection before by collecting about 5 to 10 microliters of blood from tail end and utilizing saccharometer (Glucometer Elite, BayerCorporation, Mishawaka IN) measures the tail vein glucose level.Dextrose equivalent is as time T=0.Weigh mouse and of t=0 with the oral dose of about per kilogram of body weight 2 grams or by the intraperitoneal injection glucose administration.By be used for the identical method of the method for Fundamentals of Measurement (T=0) blood glucose and after injection, measured plasma glucose concentration in about 15,30,60,90 and 120 minutes.

Mouse is put back to and allows an about week of ad libitum access in the cage, repeats GTT thereafter.Dextrose equivalent to twice check averages to be used for statistical study.Utilize Student t check to establish (pair-wise) statistical significance of paired mode.Statistical significance is defined as P＜0.05.

Embodiment 4. biological datas: insulin inhibition test (IST)

As among the above GTT, when t=0, measure the gentle body weight of G/W of tail vein.For the male mice of diet (or high fat diet), with about per kilogram of body weight 0.5 or 0.7 unit by the intraperitoneal injection administration of insulin (Humulin R, Eli Lilly and Company, Indianapolis, IN).Under rare cases, when using female mice, use the insulin of per kilogram of body weight 0.5 unit.Behind injection of insulin, measure plasma glucose levels in about 15,30,60,90 and 120 minutes and be expressed as the number percent of basic glucose.The glucose level that obtains can be represented the sensitivity of mouse to insulin, for example some tissue response insulin and the ability of ingestion of glucose.

Embodiment 5. biological datas: the insulin secretion test (GSIST) that glucose stimulates

Serum insulin level when taking the tail vein sample before the test with measurement T=0.Oral or with about per kilogram mouse body weight 2 grams by the intraperitoneal injection glucose administration.After the glucose load, collected the tail vein sample subsequently in about 7.5,15,30 and 60 minutes.By ELISA kit measurement serum insulin level (Crystal Chem Inc., Chicago, Il).

Respiratory chamber

(Columbus Instruments, Columbus Ohio) settle separately mouse in respiratory chamber.In about 48 hours a period of time, monitor metabolic rate (VO2/Kg/hr), respiratory exchange rate (RER=VC02/V02), walk about/motor activity and food and water absorption.Per approximately 48 minutes record data.Mouse is about 18 hours of overnight fasting and to observe mouse the bulimia nerovsa of overnight fast is replied approximately collecting identical data in next 24 hours subsequently.

Densitometry

(Madison WI) analyzes body fat and forms and bone mineral density (BMD) for Piximus, GEMedical Systems Lunar by DEXA (double energy X-ray absorption measurement method) densitometer.

Ptomatopsia

Collect blood by cardiac puncture, be used for the standard serum chemistry and the serum levels of leptin measured by ELISA.Weigh mesenteric mesaraic, epididymis, inguinal respectively and the brown fat pad with the assessment Fat Distribution.Gather pancreas, liver and kidney and be used for histologic analysis.

In case the behavior of Gpr100 acceptor gene deficiency is different when comparing with wild-type mice in above-mentioned test, then supports the effect of Gpr100 gene in diabetes and glucose tolerance.

Embodiment 6. biological datas: in time blood glucose horizontal survey during the fasting

Mutant and wild type animal fasting 16h and utilize OneTouch Ultra saccharometer (LifeScan) to take the tail vein sample.

That Fig. 5 shows is male (for mutant and wild type, n=7) and female (for mutant and wild type, blood glucose level n=7).This figure shows that the mutant of two kinds of sexes when the age of being checked all has the blood glucose of obvious reduction.

Embodiment 7. biological datas: in time blood glucose and insulin level measured during the fasting

The basis blood glucose (utilizes OneTouch Ultra Glucometer, LifeSpan) takes from the wild type buck (n=5) of mutant and age-matched.Remove food subsequently, move on to animal in the clean cage and per hour measure glucose.

Result (Fig. 6) show the blood glucose level of mutant between fasting 5 hours to 6 hours compare with wild type significantly drop to lower.This may be because the capability defect that animal changes to FAO after the glycogen storage depletion.

Mutant and wild type with n=6 repeat the experiment (referring to Fig. 7) that fasting is tested.Detect initial blood glucose sample once more and take blood sample (about 50 μ l) to be used for the basal insulin measurement from afterbody simultaneously.Remove food subsequently, animal moves on in the clean cage and per hour measures glucose.Taking blood sample to be used for insulin from the tail vein in the time of 6 hours measures; This sample and basic sample at room temperature condense 30 minutes subsequently 10, centrifugal 5 minutes of 000rpm.Remove serum deprivation and be stored in-80 ℃ until further analysis.

Fasting extends to 12h in this experiment.Experiment utilizes EDTA as anticoagulant when finishing, from contacting excessive CO ₂The animal vena cave take the tip blood sample, remove 50 these samples of μ l and be used for insulin and measure, it is centrifugal according to above-mentioned parameter and serum that obtain is stored in-80 ℃ until further analysis.Remaining whole blood adds Aprotinin to final concentration 500KIU/ml, and is as above centrifugal subsequently, removes serum deprivation and is stored in-80 ℃ until further analysis.

The result is presented among Fig. 7 and Figure 13.These experiments show removes after the food 6 hours, the blood glucose level of mutant animal compare with wild type significantly drop to lower, and insulin level in mutant than in wild type, improving manyly, show that mutant is hyperinsulinar.

Embodiment 8. biological datas: in time the measurement of hyperglycemic factor level during the fasting

According to product description, utilize Glucagon RIA (Linco) in above-mentioned tip sample, to measure the hyperglycemic factor level.

As shown in Figure 8, indifference between mutant and the wild type.Therefore reduce the raising that mutant when existing does not show hyperglycemic factor when glucose level, described raising is expected usually and is occurred in glycopenia state.This causes hypothesis, in case that is: the glycogen supply exhausts, animal can not change to fatty acid oxidation.

Embodiment 9. biological datas: glucose/insulin tolerance test

Measure glucose tolerance and insulin secretion after in overnight fast (16 hours) mouse, using 2mg/g (dosage/g body weight) glucose intraperitoneal injection.Take 50 μ l blood samples to be used for the insulin measurement with OneTouch Glucometer (LifeScan) Fundamentals of Measurement blood sugar and from afterbody.This sample at room temperature condensed 30 minutes, subsequently as preceding centrifugal.Every kind of animal (n=7) excites with glucose and carried out glucose measurement subsequently in 15,30,60 and 120 minutes after injection subsequently.Inject and taked another blood sample to be used for insulin from afterbody in back 60 minutes to measure; This utilizes the method preparation identical with the method that is used for the background sample.Test when finishing such as the above described tip blood sample of taking of fasting in 12 hours test that is used for.Referring to Guerre-Millo, M., et al. (2001) " PPAR-α-null mice are protected from high-fat diet-inducedinsulin resistance. " Diabetes50:2809-2814.

The result is presented among Fig. 9, Figure 10 and Figure 12.

The mutant animal has lower blood sugar level when the time is zero, based on result before, this is expected in these animals after the 16h fasting and can exists, and this lower set point means the GTT area tracing analysis under different statistically (data do not show).Yet the mutant animal does not have difference to replying with the wild type animal of exciting of glucose when data are standardized to change with respect to the basis.

The RIA of hyperglycemic factor level in the tip blood sample (as above measuring) the analysis showed that the hyperglycemic factor level of mutant does not obviously change.The result shows in Figure 11.

The elisa assay of insulin level shows that insulin level reaches higher level in mutant than in the wild type animal, shows that mutant is hyperinsulinar.

In a word, Gpr100 mutant animal presents serious hypoglycemia behind the metabolic stress of overnight fast, and this becomes obvious 6 hours the time after depriving food.This mutant has the normal tolerance of glucose and does not present the remarkable change of hyperglycemic factor level.Integrate, these results suggest mutant animal dis are changing to be used for the defective that energy produces ability to fatty acid oxidation.

List of references: Steneberg, P., et al. (2005) " The FFA receptor GPR40 linkshyperinsulinemia, hepatic steatosis, and impaired glucose homeostasis inmouse. " Cell Metabolism1:245-258.

All publications of mentioning in the above specification are introduced herein by reference. The difference of method and system described in the invention is improved and changed is apparent for a person skilled in the art, without departing from the scope and spirit of the present invention. Although the present invention interrelates with concrete preferred embodiment and is described, should understand will should be limited to undeservedly such as the present invention for required protection as described in specific embodiment. In fact, for molecular biology or various equivalent modifications significantly, the difference that is used for implementing described mode of the present invention improves and should be included within the scope of following claim.

SEQ ID NO:1 shows the cDNA sequence of people Gpr100.

GAGAAGCACTTAATTCTACAGCCTCCTTCCTAGAGCCTTCAGTGGCCTCTGCCAGTCTGGCAGACACTTGCAGACCTCTCTTCTCAGCACCACCAATCTCTGATGCCCTGCGATGCCCACACTCAATACTTCTGCCTCTCCACCCACATTCTTCTGGGCCAATGCCTCCGGAGGCAGTGTGCTGAGTGCTGATGATGCTCCGATGCCTGTCAAATTCCTAGCCCTGAGGCTCATGGTTGCCCTGGCCTATGGGCTTGTGGGGGCCATTGGCTTGCTGGGAAATTTGGCGGTGCTGTGGGTACTGAGTAACTGTGCCCGGAGAGCCCCTGGCCCACCTTCAGACACCTTCGTCTTCAACCTGGCTCTGGCGGACCTGGGACTGGCACTCACTCTCCCCTTTTGGGCAGCCGAGTCGGCACTGGACTTTCACTGGCCCTTCGGAGGTGCCCTCTGCAAGATGGTTCTGACGGCCACTGTCCTCAACGTCTATGCCAGCATCTTCCTCATCACAGCGCTGAGCGTTGCTCGCTACTGGGTGGTGGCCATGGCTGCGGGGCCAGGCACCCACCTCTCACTCTTCTGGGCCCGAATAGCCACCCTGGCAGTGTGGGCGGCGGCTGCCCTGGTGACGGTGCCCACAGCTGTCTTCGGGGTGGAGGGTGAGGTGTGTGGTGTGCGCCTTTGCCTGCTGCGTTTCCCCAGCAGGTACTGGCTGGGGGCCTACCAGCTGCAGAGGGTGGTGCTGGCTTTCATGGTGCCCTTGGGCGTCATCACCACCAGCTACCTGCTGCTGCTGGCCTTCCTGCAGCGGCGGCAACGGCGGCGGCAGGACAGCAGGGTCGTGGCCCGCTCTGTCCGCATCCTGGTGGCTTCCTTCTTCCTCTGCTGGTTTCCCAACCATGTGGTCACTCTCTGGGGTGTCCTGGTGAAGTTTGACCTGGTGCCCTGGAACAGTACTTTCTATACTATCCAGACGTATGTCTTCCCTGTCACTACTTGCTTGGCACACAGCAATAGCTGCCTCAACCCTGTGCTGTACTGTCTCCTGAGGCGGGAGCCCCGGCAGGCTCTGGCAGGCACCTTCAGGGATCTGCGGTTGAGGCTGTGGCCCCAGGGCGGAGGCTGGGTGCAACAGGTGGCCCTAAAGCAGGTAGGCAGGCGGTGGGTCGCAAGCAACCCCCGGGAGAGCCGCCCTTCTACCCTGCTCACCAACCTGGACAGAGGGACACCCGGGTGA

SEQ ID NO:2 shows the open read frame that is derived from SEQ ID NO:1.

ATGCCCACACTCAATACTTCTGCCTCTCCACCCACATTCTTCTGGGCCAATGCCTCCGGAGGCAGTGTGCTGAGTGCTGATGATGCTCCGATGCCTGTCAAATTCCTAGCCCTGAGGCTCATGGTTGCCCTGGCCTATGGGCTTGTGGGGGCCATTGGCTTGCTGGGAAATTTGGCGGTGCTGTGGGTACTGAGTAACTGTGCCCGGAGAGCCCCTGGCCCACCTTCAGACACCTTCGTCTTCAACCTGGCTCTGGCGGACCTGGGACTGGCACTCACTCTCCCCTTTTGGGCAGCCGAGTCGGCACTGGACTTTCACTGGCCCTTCGGAGGTGCCCTCTGCAAGATGGTTCTGACGGCCACTGTCCTCAACGTCTATGCCAGCATCTTCCTCATCACAGCGCTGAGCGTTGCTCGCTACTGGGTGGTGGCCATGGCTGCGGGGCCAGGCACCCACCTCTCACTCTTCTGGGCCCGAATAGCCACCCTGGCAGTGTGGGCGGCGGCTGCCCTGGTGACGGTGCCCACAGCTGTCTTCGGGGTGGAGGGTGAGGTGTGTGGTGTGCGCCTTTGCCTGCTGCGTTTCCCCAGCAGGTACTGGCTGGGGGCCTACCAGCTGCAGAGGGTGGTGCTGGCTTTCATGGTGCCCTTGGGCGTCATCACCACCAGCTACCTGCTGCTGCTGGCCTTCCTGCAGCGGCGGCAACGGCGGCGGCAGGACAGCAGGGTCGTGGCCCGCTCTGTCCGCATCCTGGTGGCTTCCTTCTTCCTCTGCTGGTTTCCCAACCATGTGGTCACTCTCTGGGGTGTCCTGGTGAAGTTTGACCTGGTGCCCTGGAACAGTACTTTCTATACTATCCAGACGTATGTCTTCCCTGTCACTACTTGCTTGGCACACAGCAATAGCTGCCTCAACCCTGTGCTGTACTGTCTCCTGAGGCGGGAGCCCCGGCAGGCTCTGGCAGGCACCTTCAGGGATCTGCGGTTGAGGCTGTGGCCCCAGGGCGGAGGCTGGGTGCAACAGGTGGCCCTAAAGCAGGTAGGCAGGCGGTGGGTCGCAAGCAACCCCCGGGAGAGCCGCCCTTCTACCCTGCTCACCAACCTGGACAGAGGGACACCCGGGTGA

SEQ ID NO:3 shows the amino acid sequence of people Gpr100.

MPTLNTSASPPTFFWANASGGSVLSADDAPMPVKFLALRLMVALAYGLVGAIGLLGNLAVLWVLSNCARRAPGPPSDTFVFNLALADLGLALTLPFWAAESALDFHWPFGGALCKMVLTATVLNVYASIFLITALSVARYWVVAMAAGPGTHLSLFWARIATLAVWAAAALVTVPTAVFGVEGEVCGVRLCLLRFPSRYWLGAYQLQRVVLAFMVPLGVITTSYLLLLAFLQRRQRRRQDSRVVARSVRILVASFFLCWFPNHVVTLWGVLVKFDLVPWNSTFYTIQTYVFPVTTCLAHSNSCLNPVLYCLLRREPRQALAGTFRDLRLRLWPQGGGWVQQVALKQVGRRWVASNPRESRPSTLLTNLDRGTPGZ

SEQ ID NO:4 shows the open read frame of the cDNA of mouse Gpr100.

TAGACCAACACCCAGATTCCAAGGGCTCTTCTAAGAGCTCTCCTGAGACAACAGCGGCGGCGGGTGGTTGCTTGCCAGGCCGGAAGGCGGGCACTCCCTGGTTCCTCTGCTCTGCTGTGCTCTAGCAACCTCCGCGGTCTTGCGATGGCCACATCCAATTCTTCTGCCTCTCTGCCCACCCTCTTCTGGGTCAATGGCTCTGGAGACAGCGTGCTGAGCACTGACGGTGCTGCCATGCCTGTCCAGTTCCTTGTTCTGAGGATCATGGTTGCACTGGCCTATGGACTTGTAGGTATCATTGGCTTGCTGGGAAATTTGGCCGTACTGTGGGTTCTAGGTAACTGTGGTCAGCGTGTGCCCGGCCTGTCTTCTGATACCTTTGTCTTCAGCCTGGCTCTAGCAGACTTGGGGCTGGCCCTTACTCTCCCTTTCTGGGCAACCGAGTCAGCAATGGACTTCCACTGGCCTTTCGGAAGTGCCCTCTGCAAGGTAGTCCTGACCACCACCGTCCTCAGCATCTATGCCAGCACCTTCCTAATCACAGCACTGAGTATCGCGCGATACTGGGTGGTAGCCATGGCTGTGGGACCAGGTAGTCACCTCTCAGTCTTTTGGGCCCGTGTGGTCACCCTGGCAGTGTGGGTGGCAGCTGCCCTGGTGACTGTGCCCACAGCAATCTTTGGGGCTGAAGTTGAGTTGTGGGGCGTGTGCCTCTGTCTTCTGCGTTTCCCCAGCAGATACTGGCTGGGAGCTTACCAGCTACAGAGGGTAGTTCTGGCCTTCATCGTGCCCTTGGGAGTCATTACCACCAGTTACCTGCTGCTGTTGGCCTTTCTAGAGCGGCAGCAAAGATGCAGGCCACGACAATGGCAGGACAGCCGAGTGGTAGCCCGCTCTGTCCGTGTCCTGGTGGCTTCCTTCGCCCTCTGCTGGGTTCCCAACCATGTAGTCACTCTCTGGGAAATTCTGGTAAGGTTTGACCTGGTGCCCTGGGACAGTACTTTCTACACCTTTCATACTTACATCCTTCCCATCACCACCTGCTTGGCCCACAGCAACAGCTGCCTCAACCCTGTGATCTATTGTCTCCTGCGGCGGGAGCCCCAGCAGGTTCTTGTCAGCTCCTTCAGAGCTCTCTGGTCAAGACTGTGGCCTCAAAGGAAGGCCTGCATGGAACAAATGGCCCTCAAGGAGGTAGGCGGGAGAACGGTAGCCAGCACCCAGGAGAGTGGCTCTTCTAGGACACACACAAACACAATGGAACACCTGGATGAAGGATGCAGCCTGAACACTCTCCTTTCTGAGACCTATCAGGGGCAGAGCCCACAGATTCTAGGGAGGAGCAGCTGCTCTCTCAGTCAGGCTGCTGTGTCCCCAGGAGAAGTCTGA

SEQ ID NO:5 shows the amino acid sequence of mouse Gpr100.

MATSNSSASLPTLFWVNGSGDSVLSTDGAAMPVQFLVLRIMVALAYGLVGIIGLLGNLAVLWVLGNCGQRVPGLSSDTFVFSLALADLGLALTLPFWATESAMDFHWPFGSALCKVVLTTTVLSIYASTFLITALSIARYWVVAMAVGPGSHLSVFWARVVTLAVWVAAALVTVPTAIFGAEVELWGVCLCLLRFPSRYWLGAYQLQRVVLAFIVPLGVITTSYLLLLAFLERQQRCRPRQWQDSRVVARSVRVLVASFALCWVPNHVVTLWEILVRFDLVPWDSTFYTFHTYILPITTCLAHSNSCLNPVIYCLLRREPQQVLVSSFRALWSRLWPQRKACMEQMALKEVGGRTVASTQESGSSRTHTNTMEHLDEGCSLNTLLSETYQGQSPQILGRSSCSLSQAAVSPGEVZ

TTGTGCAGAGTTCAATGGAGAATGTTG-SEQ ID No.6

CCAGAAACACTCTACGCCTGTCACCTG-SEQ ID No.7

TttgcggccgcAAAGTGACTCATGCTGCTCCCATCTTC-SEQ ID No.8

AaaactagTCCCAGCAAGCCAATGATACCTACAAG-SEQ ID No.9

TttggcgcgCCTGGGACAGTACTTTCTACACCTTTC-SEQ ID No.10

TttggccggccTCCATTTAAGAAGAGATCTTGAGCCAG-SEQ ID No.11

TGGATCCTTTTATTTTGGAGACTGAAC-SEQ ID No.12

CCTGGCTCAAGATCTCTTCTTAAATGG-SEQ ID No.13

GGTGAGCAATCAGATCATGAGACTTAC-SEQ ID No.14

GCTTACCAGCTACAGAGGGTAGTTCTG-SEQ ID No.15

TGATGGGAAGGATGTAAGTATGAAAGGTG-SEQ ID No.16

GTCGTGACCCATGGCGATGCCTGCTTG-SEQ ID No.17

CGGATCCACTAGATAACTTCGTATAGC-SEQ ID No.18

SEQ ID No.19: from the genomic gene seat of 5 ' prF to 3 ' prR

Sequence scope: 25401 to 32500

> 5’prF

|

| 25500

GTGTGTGTTTGTGTGCACTTATGCATTTGTGCAGAGTTCAATGGAGAATGTTGGGTGATAGTCTTCACTAGCCACCTTAAGATGGTCTTCTTGTTCACCC

CACACACAAACACACGTGAATACGTAAACACGTCTCAAGTTACCTCTTACAACCCACTATCAGAAGTGATCGGTGGAATTCTACCAGAAGAACAAGTGGG

25600

ATAGGTGACCCATGAGTYYCCCAGGTCCAACTATGTAGTGGTTTAAATAAGTATTTGCCCGTAGGCTCAGATATTTAAACACTGGGAGTTCAGTTAGTGA

TATCCACTGGGTACTCAAAGGGTCCAGGTTGATACATCACCAAATTTATTCATAAACGGGCATCCGAGTCTATAAATTTGTGACCCTCAAGTCAATCACT

<5’prR

|

| 25700

CCCTAACTGTGCTGTTTGGGGAGGTTGGAAACCTTTGACAGGTGGAGCCTTGTTAGAAGAATGTCTCCAGGTGACAGGCGTAGAGTGTTTCTGGTCTTGC

GGGATTGACACGACAAACCCCTCCAACCTTTGGAAACTGTCCACCTCGGAACAATCTTCTTACAGAGGTCCACTGTCCGCATCTCACAAAGACCAGAACG

25800

CTCACTTCCTGCTCTCTTAGTGCCAAGCTCTTGTTACTGTGCCTGCTGCCCTGCCTCCATTCTTCCCCACCATGGTGGACTCTAGCCTTCAGGAACCATA

GAGTGAAGGACGAGAGAATCACGGTTCGAGAACAATGACACGGACGACGGGACGGAGGTAAGAAGGGGTGGTACCACCTGAGATCGGAAGTCCTTGGTAT

25900

AGCCGAAAGAAACTTCCTTAATTAAGTTGCCTTGGCCATGGCATTTTCTCAGAGCAACAGAAAAGTGTAGTGAGAATTTTGGTTTCTTTAAAAACCACAT

TCGGCTTTCTTTGAAGGAATTAATTCAACGGAACCGGTACCGTAAAAGAGTCTCGTTGTCTTTTCACATCACTCTTAAAACCAAAGAAATTTTTGGTGTA

26000

GACGGCCGGGCATGGTGGCGCATGCCTTTAATCCCAGCACTCGGGAGGCAGAGGCAGGCGGATTTCTGAGTTTGAGGCCAGCCTGGTCTACAAAGTGAGC

CTGCCGGCCCGTACCACCGCGTACGGAAATTAGGGTCGTGAGCCCTCCGTCTCCGTCCGCCTAAAGACTCAAACTCCGGTCGGACCAGATGTTTCACTCG

26100

TCCAGGACAGCCAGGGCTATACAGAGAAACCATGTCTCGAAAAAACAAACAAACAAACAAAAACAAAAAACAAAAAAACCACAGAACTAGGAATGTGCTT

AGGTCCTGTCGGTCCCGATATGTCTCTTTGGTACAGAGCTTTTTTGTTTGTTTGTTTGTTTTTGTTTTTTGTTTTTTTGGTGTCTTGATCCTTACACGAA

26200

CTGTTTTGATCCTAGGTGTGGGGACACGGGGCTGCTTCAGATTGAGCACAGCAGCTGACCTTGGCTTGCCCCGTGCTCTAACAGCAGTGTGACTTTCCCA

GACAAAACTAGGATCCACACCCCTGTGCCCCGACGAAGTCTAACTCGTGTCGTCGACTGGAACCGAACGGGGCACGAGATTGTCCTCACACTGAAAGGGT

26300

GTTACAGATAGTTTTTGTGACTGTGTGACAATTGGGATGCTGGGGACTTTTCAGAGGGTCTATAAATGCTAGGGCCCCAAGAGGGAGCATGGGTTGTTGG

CAATGTCTATCAAAAACACTGACACACTGTTAACCCTACGACCCCTGAAAAGTCTCCCACATATTTACGATCCCGGGGTTCTCCCTCGTACCCAACAACC

26400

TTGCTGTGAATTGTTGTCAGTCTCCATTTGTTAAGTGGTTGTGTGCAAAGAAGAAGCAAGAAAAAGAAATTAGATATCCTGATGGCAAAGATCAAACTTG

AACGACACTTAACAACAGTCAGAGGTAAACAATTCACCAACACACGTTTCTTCTTCGTTCTTTTTCTTTAATCTATAGGACTACCGTTTCTAGTTTGAAC

26500

CCTCAAGGAACTCAATGCTCCTAAGAAGGGAGTGGCCTAACAATAACATCGCCCCTTCCCGACCCCTGAATTTACCCAGGAATCTCTTTCCTTTCCTCTC

GGAGTTCCTTGAGTTACGAGGATTCTTCCCTCACCGGATTGTTATTGTAGCGGGGAAGGGCTGGGGACTTAAATGGGTCCTTAGAGAAAGGAAAGGAGAG

>5’armF

|

26600

TGTCCCCTTTTCTCTTCTATCTAATGCTGCAAGAAAAGGGATGGAGAAGGGTGGAAGAAAGAAAAACTCACAACGTAGCCCAAGTCCTGCTACAGAAAAG

ACAGGGGAAAAGAGAAGATAGATTACGACGTTCTTTTCCCTACCYCTTCCCACCTTCTTTCTTTTTGAGTGTTGCATCGGGTTCAGGACGATGTCTTTTC

26700

TGACTCATGCTGCTCCCATCTTCCTGGGAGTCCCAGGGTTACAGGTGCTCTCTGCTGAGCACAGCTCTGCCTGCATAAGCTCTAGGCCTCTGAATGGGGA

ACTGAGTACGACGAGGGTAGAAGGACCCTCAGGGTCCCAATGTCCACGAGAGACGACTCGTGTCGAGACGGACGTATTCGAGATCCGGAGACTTACCCCT

26800

CCCTCATGCATGCATCTTATCTCCTCAGTAAACTCCCTCTCCAACCTAAGCTGCATGGTTTTGGTTTTATTCTGTGTGCAAGTGATGGGGTTGGAGGATT

GGGAGTACGTACGTAGAATAGAGGAGTCATTTGAGGGAGAGGTTGGATTCGACGTACCAAAACCAAAATAAGACACACGTTCACTACCCCAACCTCCTAA

26900

GCAGCAAGCAAGGGAGCAGTGTTCTGCTGATCTACACCGGCAGCCGGGTCGCTGGAGTGCCTAAGCTGGCTATACAATTCTGGCCAAAACAGCATGGCCG

CGTCGTTCGTTCCCTCGTCACAAGACGACTAGATGTGGCCGTCGGCCCAGCGACCTCACGGATTCGACCGATATGTTAAGACCGGTTTTGTCGTACCGGC

27000

TAAGAAATGAATACTTGACATGAGTCGGACTCAGACTATGACGTCAGGAAGACCCCTCCGCGCACAAGCTCAGGCTGTGAAATCTCACCAACCCCCACCC

ATTCTTTACTTATGAACTGTACTCAGCCTGAGTCTGATACTGCAGTCCTTCTGGGGAGGCCCGTGTTCGAGTCCGACACTTTAGACTGGTTGGGGGTGGG

27100

TGGAAAACTCTGCCCCACAAGAAAAGCTGTATAACACGTGTCTTCTGCTCAGTTCTCTGTAGCTTCTCTCCCTAGCTGAGGTGTCTTTCCCAATAAATCT

ACCTTTTGAGACGGGGTGTTCTTTTCGACATATTGTGCACAGAAGACGAGTCAAGAGACATCGAAGAGAGGGATCGACTCCACAGAAAGGGTTATTTAGA

27200

ATTGTGTGGGGTTTGTTGTGCCGAGTGACTTTGTCCTATTATTCCTTGACTCCTGACTGCTAGGACACCTTTCTCTTCACAGCTATAACACAGGTAGCCC

TAACACACCCCAAACAACACGGCTCACTGAAACACGATAATAAGGAACTGAGGACTGACGATCCTGTGGAAAGAGAAGTGTCGATATTGTGTCCATCGGG

27300

TTGGGTTGCAGCTGAGTGCCTTCTGCCAACCTCATCCCACCTCTCTCTTGCTCAAAACCCCAACATTCTCTTTCCCAGAAAGCAACTCTCTCACGTCTGG

AACCCAACGTCGACTCACGGAAGACGGTTGGAGTAGGGTGGAGAGAGAACGAGTTTTGGGGTTGTAAGACAAAGGGTCTTTCGTTGAGAGAGTGCAGACC

27400

GGTTCTCATATGGGCTAGAGCTTCTCTGAAATGCCTCTCTGACCCCACCCCCAGCACCCCAAGACAACCGAGTACCAGATACTTCCTTAACCAGCAGAAG

CCAAGAGTATACCCGATCTCGAAGAGACTTTACGGAGAGACTGGGGTGGGGGTCGTGGGGTTCTGTTGGCTCATGGTCTATGAAGGAATTGGTCGTCTTC

27500

AGTGCCGAACTGCTCTCAGAATCCTTGAAAAAAAAATCTCACTCTTACCTCCCCACTCCATGCTCTCTCCCAAAGCTCCTGCCGTGGAGAGAGATGTGAG

TCACGGCTTCACGAGAGTCTTAGGAACTTTTTTTTTAGAGTGAGAATGGAGGGGTCAGGTACGAGAGAGGGTTTCGAGGACGGCACCTCTCTCTACACTC

27600

GAGAGAAAGGAGATGATGCCATTAGAGACGGTCTTCAGCTGAGACTGAGCTCATGGGGAGACAGGATGAGGTGTGGTAAAGGCTTTTCTCAGGTTCCAGA

CTCTCTTTCCTCTACTACGGTAATCTCTGCCAGAAGTCGACTCTGACTCGAGTACCCCTCTGTCCTACTCCACACCATTTCCGAAAAGAGTCCAAGGTCT

27700

ACTCTACAGTGAGGCTTGGGATATGGAGGAATGTGGATATGACGGGGCTCTGAGTACACCAGAAAAATGGAGTGGCAGTGGGGGTGGGACAGGTTGGCAG

TGAGATGTCACTCCGAACCCTATACCTCCTTACACCTATACTGCCCCGAGACTCATGTGGTCTTTTTACCTCACCGTCACCCCCACCCTGTCCAACCGTC

27800

GACTGTTCAGGGATGTAGAGCAGACCTGAAGGTGTGGCCGTGGTCATCCACCTATGGCCTCACAAGTCCAATGTGCCCAGAAGGGGTATACAGGCAAGTG

CTGACAAGTCCCTACATCTCGTCTGGACTTCCACACCGGCACCAGTAGGTGGATACCGGAGTGTTCAGGTTACACGGGTCTTCCCCATATGTCCCTTGAC

27900

CAACTTCCTCCGTCTCATTTCTTTCCCCTTCTTAGCATCAAGGTCACATGGTACTGCACTGAATTCTGACCTGTGTTTTAGTTGGCAGTGTTCCCTTGGC

GTTGAAGGAGGCAGAGTAAAGAAAGGGGAAGAATCGTAGTTCCAGTGTACCATGACGTGACTTAAGACTGGACACAAAATCAACCGTCACAAGGGAACCG

28000

ATGATAACAGCTGTTCATTGTCCTGCTTCCTTCCATGACCAATGCTCCAAACTTCCCCTTGAAATGACAGTCATCATGCAAAGAACAAGGTATGGACTCA

TACTATTGTCGACAAGTAACAGGACGAAGGAAGGTACTGGTTACGAGGTTTGAAGGGGAACTTTACTCTCAGTAGTACGTTTCTTGTTCCATACCTGAGT

28100

CGCTAGCTCCCTGAACAACGGACTATCTTCATCTGATTTAACTTCTGTAAGGAATTTTTATTTATATGTATGTCTGCACGTATGGACATGTAGCACATTC

GCGATCGAGGGACTTGTTGCCTGATAGAAGTAGACTAAATTGAAGACATTCCTTAAAAATAAATATACATACAGACGTGCATACCTGTACATCGTGTAAG

28200

GAGCCAGTGGAGACCAAAAGAGGGGGTCAAATTCTTTAGAACTGGAGTGATAGATGACGGTGAGATACTAGATGGGTGCTGGGTACCACACGCAGGTCAT

CTCGGTCACCTCTGGTTTTCTCCCCCAGTTTAAGAAATCTTGACCTCACTATCTACTGCCACTCTATGATCTACCCACGACCCATGGTGTGCGTCCAGTA

28300

CTGCAAGATACTAACTCCTTAGGTATCTCTAGCCCCAATTTAAAACTATTTAAATAGTATTTGCCTGTGTGAGCATGGATGTTCAGTATGCTACTGAATG

GACGTTCTATGATTGAGGAATCCATAGAGATCGGGGTTAAATTTTGATAAATTTATCATAAACGGACACACTCGTACCTACAAGTCATACGATGACTTAC

28400

CAGGCAAATGTCAGGACAACTTTCAGGAATCTGTTCTCTCCTCCCACCTTGTGGATCCTGACCACCCAGTGCACATTGTCAGGGCCGACAAGCAGGTACT

GTCCGTTTACAGTCCTGTTGAAAGTCCTTAGACAAGAGAGGAGGGTGGAACACCTAGGACTGGTGGGTCACGTGTAACAGTCCCGGCTGTTCGTCCATGA

28500

TGATATCTAGTCCCAAACTTGTTTGGTTTGGTTTTGAGACAGGGTTTCAGAACCTAACCTTGTCTGGCCTGGAATTCAGTAGACAGACCAGGCTGGGTGT

ACTATAGATCAGGGTTTGAACAAACCAAACCAAAACTCTGTCCCAAAGTCTTGGATTGGAACAGACCGGACCTTAAGTCATCTGTCTGGTCCGACCCACA

28600

GCCAGCAAACCATTAAGCCTGTCACCATCTAATCTTATTTTGAAGATTTAGTAGTGCTGGGGGGTACATACTACTGTGCATCTATAAAAGTCAGAAGACA

CGGTCGTTTGGTAATTCGGACAGTGGTAGATTAGAATAAAACTTCTAAATCATCACGACCCCCCATGTATGATGACACGTACATATTTTCAGTCTCTTGT

28700

ACCCCGGGATGCCTGTCCTCTCCTACCTTTATGTAGGCAGGTTCCAGGGACAGCCATCTCCACGGGCGTATTTATTTATATATAGATTATACCTTGTTGT

TGGGGCCCTACGGACAGGAGAGGATGGAAATACATCCGTCCAAGGTCCCTCTCGCTAGAGGTGCCCGCATAAATAAATATATATCTAATATGGAACAACA

28800

ACTGCCAAGTTTGGCCTTGGTGATCAGGTGATCCAGCTGCCTCAGCCATGCAGGTAGCAGTGACCACAGCTCTTTGGCATCTCGCTGAATTGTATATTTT

TGACGGTTCAAACCGGAACCACTAGTCCACTAGTCGACGGAGTCGGTCACGTCCATCGTCACTGGTGTCGAGAAACCGTAGAGCGACTTAACATATAAAA

28900

CTTAACACTTTTGGGGGGAGAATTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGAGATGAGTGTGGAGGTCAAAGGGCAGCTTGTGAGT

GAATTGTGAAAACCCCCCTCTTAACACACACACACACACACACACACACACACACACACACACACTCTACTCACACCTCCAGTTTCCCGTCGAACACTCA

29000

GGCATGTCTATCCTTCTCCTATGCAGATCCTGTAGACTGAATTCAAATCACCAGGCTTGGCAGCAGGCCCCTTTCCCCAAACAGCCCCGATCTCTGGCCT

CCGTACAGATAGGAAGAGGATACGTCTAGGACATCTGACTTAAGTTTAGTGGTCCGAACCGTCGTCCGGGGAAAGGGGTTTGTCGGGGCTACAGACCGGA

29100

GCCCCCATTAACATTTTAAGCAAAGCAGCACAGGGCCCCAATCAGAGAGACACAGGAAAGGCACAGCAACTGTGGGACTCTGAGTGATGTGTGTTCCTCT

CGGGGGTAATTGTAAAATTCGTTTCGTCGTGTCCCGGGGTTAGTCTCTCTGTGTCCTTTCCCTGTCCTTGACACCCTGAGACTCACTACACACAAGGAGA

29200

CTCTTTTTCTGACTCCCAGTCTGGTTTTCTAATGCCATATTCCTTCCCTAACCTCTCCTCACACCTCTCCTTTTCTCAGAGAGCTCTCTGCACTCCGGGG

GAGAAAAAGACTGAGGGTCAGACCAAAAGATTACGGTATAAGGAAGGGATTGGAGAGGAGTGTGGAGAGGAAAAGAGTCTCTCGAGAGACGTGAGGCCCC

29300

AGAGAGAAAAAGCGTCTTCTGGTTTGGCTCCACATCTCTACTGTTTCCCTTTCCTTCCCTATTACATGCATGTTGACAGTGTGAACATAGCCCTCCCTTG

TCTCTCTTTTTCGCAGAAGACCAAACCGAGGTGTAGAGATGACAAAGGGAAAGGAAGGGATAATGTACGTACAACTGTCACACTTGTATCGGGAGGGAAC

29400

CACTGCCCCACCCACCACGGTTGTCCAACTGGGAAGAAGCAGGCTGTTCTACCCACACCTGTCCCTCTTAGGTGTGAGCATGGGCAGGGGCTCTTACAAC

GTGACGGGGTGGGTGGTGCCAACAGGTTGACCCTTCTTCGTCCGACAAGATGGGTGTGGACAGGGAGAATCCACACTCGTACCCGTCCCCGACAATGTTG

29500

CCGAAGCTCTAGACCAACACCCAGATTCCAAGGGCTCTTCTAAGAGCTCTCCTGAGACAACAGCGGCGGCGGGTGGTTGCTTGCCAGGCCGGAAGGCGGG

GGCTTCGAGATCTGGTTGTGGGTCTAAGGTTCCCGAGAAGATTCTCGAGAGGACTCTGTTGTCGCCGCCGCCCACCAACGAACGGTCCGGCCTTCCGCCC

29600

CACTCCCTGGTTCCTCTGCTCTGCTGTGCTCTAGCAACCTCCGCGCTCTTGCGATGGCCACATCCAATTCTTCTGCCTCTCTGCCCACCCTCTTCTGGGT

GTGAGGGACCAAGGAGACGAGACGACACGAGATCGTTGGAGGCGCCAGAACGCTACCGGTGTAGGTTAAGAAGACGGAGAGACGGGTGGGAGAAGACCCA

M A T S N S S A 9 L P T L F W V >

_______________________MUSGPR100C __________________ >

29700

CAATGGCTCTGGAGACAGCGTGCTGAGCACTGACGGTGCTGCCATGCCTGTCCAGTTCCTTGTTCTGAGGATCATGGTTGCACTGGCCTATGGACTTGTA

GTTACCGAGACCTCTCTGCACGACTCGTGACTGCCACGACGGTACGGACAGGTCAACGGAACAAGACTCCTAGTACCAACGTGACCGGATACCTGAACAT

N G S G D S V L S T D G A A M P V Q F L V L R I M V A L A Y G L V >

____________________________________________MUSGPR100C______________________________________________>

<5’armR

|

>7tm

| |

| | 29800

GGTATCATTGGCTTGCTGGGAAATTTGGCCGTACTGTGGCTTCTAGGTAACTGTGGTCAGCGTGTGCCCGGCCTGTCTTCTGATACCTTTGTCTTCAGCC

CCATAGTAACCGAACGACCCTTTAAACCGGCATGACACCCAAGATCCATTGACACCAGTCGCACACGGGCCGGACAGAAGACTATGGAAACACAAGTCGG

G I I G L L G N L A V L W V L G N C G Q R V P G L S S D T F V F S >

____________________________________________MUSGPR100C _____________________________________________>

29900

TGGCTCTAGCAGACTTGGGGCTGGCCCTTACTCTCCCTTTCTGGGCAACCGAGTCAGCAATGGACTTCACTGGACCTTTCGGAAGTGCCCTCTGCAAGGT

ACCGAGATCGTCTGAACCCCGACCGGGAATGAGAGGGAAAGACCCGTTGGCTCAGTCGTTACCTGAAGGTGACCGGAAAGCCTTCACGGGAGACGTTCCA

L A L A D L G L A L T L P F W A T E S A M D F H W P F G S A L C K V >

____________________________________________MUSGPR100C_______________________________________________>

30000

AGTCCTGACCACCACCGTCCTCAGCATCTATGCCAGCACCTTCCTAATCACAGCACTGAGTATCGCGCGATACTGGGTGGTAGCCATGGCTGTGGGACCA

TCAGGACTGGTGGTGGCAGGAGTCGTAGATACGGTCGTGGAAGGATTAGTGTCGTGACTCATAGCGCGCTATGACCCACCATCGGTACCGACACCCTGGT

V L T T T V L S I Y A S T F L I T A L S I A R Y W V V A M A V G P>

____________________________________________MUSGPR100C______________________________________________>

30100

CCTAGTCACCTCTCAGTCTTTTGGGCCCGTGTGGTCACCCTGGCAGTGTGGGTGGCAGCTGCCCTGGTGACTGTGCCCACAGCAATCTTTGGGGCTGAAG

CCATCAGTGGAGAGTCAGAAAACCCGGGCACACCAGTGGGACCGTCACACCCACCGTCGACGGGACCACTGACACGGGTGTCGTTAGAAACCCCGACTTC

G S H L S V F W A R V V T L A V W V A A A L V T V P T A I F G A E>

___________________________________________MUSGPR100C_______________________________________________>

>hetF

|

| 30200

TTGAGTTGTGGGGCGTGTGCCTCTGTCTTCTGCGTTTCCCCAGCAGATACTGGCTGGCAGCTTACCAGCTACAGAGGGTAGTTCTGGCCTTCATCCTGCC

AACTCAACACCCCGCACACGGAGACAGAAGACGCAAAGGGGTCGTCTATGACCGACCCTCGAATGGTCGATGTCTCCCATCAAGACCGGAAGTAGCACGG

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

____________________________________________MUSGPR100C______________________________________________>

30300

CTTGGGAGTCATTACCACCAGTTACCTGCTGCTGTTGGCCTTTCTAGAGCGGCAGCAAAGATGCAGGCCACGACAATGGCAGGACAGCCGAGTGGTAGCC

GAACCCTCAGTAATGGTGGTCAATGGACGACGACAACGGAAAGATCTCGCCGTCGTTTCTACGTCCGGTGCTGTTTACCGTCCTGTCGGCTCACCATCGG

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

____________________________________________MUSGPR100C _____________________________________________>

>3’arm

|

>3’armF

|

30400

CGCTCTGTCCGTGTCCTGGTGGCTTCCTTCGCCCTCTGCTGGGTTCCCAACCATGTAGTCACTCTCTGGGAAATTCTGGTAAGGTTTGACCTGGTGCCCT

GCGAGACAGGCACAGGACCACCGAAGGAAGCGGGAGACGACCCAAGGGTTGGTACATCAGTGAGAGACCCTTTAAGACCATTCCAAACTGGACCACGGGA

R S V R V L V A S F A L C W V P N H V V T L W E I L V R F D L V P>

____________________________________________MUSGPR100C______________________________________________>

<hetR

|

| 30500

GGGACAGTACTTTCTACACCCCTTTCATACTTACATCCTTCCCATCACCACCTGCTTGGCCCACAGCAACAGCTGCCTCAACCCTGTGATATTGTCTCCT

CCCTCTCATGAAAGATGTGGAAAGTATGAATGTAGGAAGGGTAGTGGTGGACGAACCGGGTGTCGTTGTCGACGGAGTTGGGACACTAGATAACAGAGGA

W D S T F Y T F H T Y I L P I T T C L A H S N S C L N P V I Y C L L>

____________________________________________MUSGPR100C_______________________________________________>

30600

GCGGCGGGAGCCCCAGCAGGTTCTTGTCAGCTCCTTCAGAGCTCTCTGGTCAAGACTGTGGCCTCAAAGGAAGGCCTGCATGGAACAAATGGCCCTCAAG

CGCCGCCCTCGGGGTCGTCCAAGAACAGTCGAGGAACTCTCGAGAGACCAGTTCTGACACCGGAGTTTCCTTCCGGACGTACCTTGTTTACCGGGAGTTC

R R E P Q Q V L V S S F R A L W S R L W P Q R K A C M E Q M A L K>

____________________________________________MUSGPR100C___ __________________________________________>

30700

GAGGTAGGCGGGAGAACGGTAGCCAGCACCCAGGAGAGTGGCTCTTCTAGGACACACACAAACACAATGGAACACCTGGATGAAGGATGCAGCCTGAACA

CTCCATCCGCCCTCTTGCCATCGGTCGTGGGTCCTCTCACCGACAAGATCCTGTGTGTGTTTGTGTTACCTTGTGGACCTACTTCCTACCTCGGACTTCT

E V G G R T V A S T Q E S G S S R T H T N T M E H L D E G C S L N>

____________________________________________MUSGPR100C_____________________________________________>

30800

CTCTCCTTTCTGAGACCTATCAGGGGCAGAGCCCACAGATTCTAGGGAGGAGCAGCTGCTCTCTCAGTCAGGCTGCTGTGTCCCCAGGAGAAGTCTGATC

GAGAGGAAAGACTCTGGATAGTCCCCGTCTCGGGTGTCTAAGATCCCTCCTCGTCGACGAGAGAGTCAGTCCGACGACACAGGGGTCGTCTTCAGACTAG

T L L S E T Y Q G Q S P Q I L G R S S C S L S Q A A V S P G E V *>

___________________________________________MUSGPR100C ____________________________________________>

30900

TTTGATCACCAACTCTGGGTGTGACAGAACGGAGAAGCTGGAGTCCAAACAGGAGGTGGATGTGGCAAACCTTATCTCTGGAGATGGCAAAGAGGAACTG

AAACTAGTGGTTGAGACCCACACTGTCTTGCCTCTTCGACCTCAGGTTTGTCCTCCACCTACACCGTTTCGAATAGAGACCTCTACCGTTTCTCCTTGAC

31000

AGAATAAACCAGATCGGTCAGAGACTGTCTTGACCTTTCATGCAAGTACTTCACAGGATAACTAACGTCCATCACCCGGTTTAGACTAACAGGTCAGGTG

TCTTATTTGGTCTAGCCAGTCTCTGACAGAACTGGAAACTACGTTCATGAAGTGTCCTATTGATTCCAGGTAGTGGGCCAAATCTGATTGTCCAGTCCAC

31100

TCGGTTCTCCCTGTCACTTTAGAATAGGGCACCCGTTATGCCTCTTTGGTACCAAACCCAAAAATGTATTCTCTGGCCAATAAGCTTTTTGTCATCTTAG

AGCCAAGAGGGACAGTGAAATCTTATCCCGTGGGCAATACGGAGAAACCATGGTTTGGGTTTTTACATAAGAGACCGGTTATTCGAAAAACAGTAGAATC

31200

AGGTACCCATTAGGAATGATGTAGAACCCTCCCCTTCAACGTTTGTCTGTCGTTTTGCTGCCACAAGATGCAGACCCTGAGTGTATAGCCTTTGAGAATA

TCCATGGGTAATCCTTACTACATCTTCGGAGGGGAAGTTGCAAACAGACACGAAAACGACGGTGTTCTACGTCTGGGACTCACATATCGGAAACTCTTAT

31300

GTGAATAGATCTGTCCCTTTCAATCAAGGATTGGGTAACAATCACAAGGGTCTGGGCGTGGGGGTGGGGACTCAAGAGATACAGAAAAGTTTTGTAGGCT

CACTTATCTAGACAGGGAAAGTTACTTCCTAACCCATTGTTAGTGTTCCCAGACCCGCACCCCCACCCCTCAGTTCTCTATGTCTTTTCAAAACATCCGA

31400

GAGGGTCAGAAACCAGAAGCTAGTCTCACTGAGTACAACTGCACTTCAGCCAAGCGCCAGAGCATGTGGGCTGGAATCTTCCCCCCACGCAAATTATTCT

CTCCCAGTCTTTGGTCTTCGATCAGAGTGACTCATGTTGACGTGAAGTCGGTTCGCGGTCTCGTACACCCGACCTTAGAAGGGGGGTCCGTTTAATAAGA

31500

AGAAGTCAGTTCTCCCCTTCTAAAATTGGAGGCAGGGTTTCATCATACCCAGGCTGGTCTCACACTGTAAAGCTGAGGGTCGCTTGGAATCTTTAATCTT

TCTTCAGTCAAGAGGGGAAGATTTTAACCTCCGTCCCAAAGTAGTATGGGTCCGACCAGAGTGTGACATTTCGACTCCCAGCGAACCTTAGAAATTAGAA

31600

GATCCCAGATGCTCGAATTATAGGCATGTGCCAACAAGTTGAGCTTTTCAGATCATTTTAGCCTATTCTTCCTTCTTTCCTGTACTTATGTATGTATGTA

CTAGGGTCTACGAGCTTAATATCCGTACACGGTTGTTCAACTCGAAAAGTCTAGTAAAATCGGATAAGAAGGAAGAAAGGACATGAATACATACATACAT

31700

TGCATGGCTTGCATGTACATTTGTGTTCACACTTCTACGTGTGTCAGAAGCAAACATAAGGTTTTTCATTTCCTCGTCTTTGTTTTTATTGAGACGGTCT

ACGTACGAACGTACATGTAAACACAAGTGTGAACATGCACACAGTCTTCGTTTGTATTCCAAAAAAGTAAAGGAGCAGAAACAAAAATAACTCTGCCAGA

31800

TACGACCTAACCCTAACTGTCCTGGAATTCACTCTGTAGACCAGGCTGTCCCTTGAACTCAGACACTCACCTGCTTCTGCCTCCCATGTGCTGGAACTAC

ATGCTGGATTGGGATTGACAGGACCTTAAGTGAGACATCTGGTCCGACAGGGAACTTGAGTCTGTGAGTGGACGAAGACGGAGGGTACACGACCTTGATG

31900

AGGCATGTGCCAACGCACCTTGCTTTGATTTTTAAAAAATTACACTTTATTCAAGTCTGTTCAGACGAGGAGACATGTTCCACATGCATGTGGGCGATCA

TCCGTACACGGTTGCGTGGAACGAAACTAAAAATTTTTTTAATGTGAAATAAGTTCACACAAGTCTGCTCCTCTGTACAAGGTGTACGTACACCGCTAGT

32000

GGACAGCTTGGGGTCTGCTTTCTCTTTCCCCCCCTCTTAAGATTCTGGAGTCAGCTTAGGCCATCAGGCTTTCGGGCAAGGGTCTTTACCCGGGGAAGCA

CCTCTCGAACCCCAGACGAAAGAGAAAGGGGGGAGAATTCTAAGACCCTCAGTCGAATCCGGTAGTCCGAAAGCCCGTTCCCAGAAATGGGCCCCTTCGT

32100

GTTTTCTAAACCCTCCACACGATGTTTATTTGGGTTTTCTTGTTTTTTTCTGAGACAGGGTTTCTCTGTGTAGCCCTGGCTGTCCTGGAGCTCACTTTGT

CAAAAGATTTGGGAGGTGTGCTACAAATAAACCCAAAAGAACAAAAAAAGACTCTGTCCCAAAGAGACACATCGGGACCGACAGGACCTCGAGTGAAACA

>3’prF

|

| 32200

AGACCAAGCTGACTTCGAATTCAGAAATCCGCCTGCCTCTGCCTCCCAAGAGCTGGGATTAAAGGCGTGCTCCACCACGCCTGGCTCAAGATCTCTTCTT

TCTGGTTCGACTGAAGCTTAAGTCTTTAGGCGGACGGAGACGGAGGGTTCTCGACCCTAATTTCCGCACGAGGTGGTGCGGACCGAGTTCTAGAGAAGAA

<3’armR <3’scr

| |

| | 32300

AAATGGAATAAAAGGTTCAGTCTCCAAAATAAAAGGATCCAGCAGTGCCATAGGCAGGGTTCCCGGTACTGACACCTTCCATGGAAAATGGAAAGACGAC

TTTACCTTATTTTCCAAGTCAGAGGTTTTATTTTCCTAGGTCGTCACGGTATCCGTCCCAAGGGCCATGACTGTGGAAGGTACCTTTTACCTTTCTCGTG

32400

AGAAAACATGGGCAGCTGGCAGACAGGTACAGGTGAGCCACTGAGGTCTCAGTGGTATCTTACATGGACTTGTTTATGGAATATAATGTAAGTCTCATGA

TCTTTTGTACCCGTCGACCGTCTGTCCATGTCCACTCGGTGACTCCAGAGTCACCATAGAATGTACCTGAACAAATACCTTATATTACATTCAGAGTACT

<3’prR

|

| 32500

TCTGATTGCTCACCCTCACGCGCGCGCCGCACATTTGTGAGAACACACACACATAATAAAGTAGAACCAGCAAGATGGCGCAGCAGGGAAAACACATGGC

AGACTAACGAGTGGGAGTGCGCGCGCGCGCGTGTAAACACTCTTGTGTGTGTATTATTTCATTCTTGGTCGTTCTACCGCGTCGTCCCTTTTGTGTACCG

Claims (22)

1. identify and be suitable for treatment, prevent or alleviate the Gpr100 relevant disease, the method of the molecule of diabetes and obesity particularly, this method comprises determines whether candidate molecules is the activator or the antagonist of Gpr100 polypeptide, wherein said Gpr100 polypeptide comprises the amino acid sequence shown in SEQ ID NO.3 or the SEQ ID NO:5, or with its at least 90% same sequence.

2. the process of claim 1 wherein described Gpr100 polypeptide by the nucleotide sequence shown in SEQ ID No.1, SEQ IDNo.2 or the SEQ ID NO:4 or with its at least 90% same sequential coding.

3. claim 1 or 2 method comprise making candidate molecules contact Gpr100 polypeptide, and detect because the variation of the intracellular Ca2+ level that contact causes.

4. claim 1 or 2 method comprise making non-human animal or its part, preferred cell, tissue or organ contact candidate molecules, and determine whether described contact causes the change of described animal organism mathematic(al) parameter.

5. the method for claim 4, wherein said biological parameter is selected from following group: serum level of glucose, body weight, hyperglycemic factor level, fatty percent.

6. have transgenic nonhuman animal or its isolated cells of the ruined endogenous Gpr100 of function or organize as glucose adjusting or Gpr100 relevant disease the purposes of the model of preferred obesity or diabetes.

7. the purposes of claim 6, wherein said transgenic nonhuman animal comprises the ruined Gpr100 gene of function, preferably comprises disappearance in Gpr100 gene or its part.

8. claim 6 or 7 purposes or method, wherein said transgenic nonhuman animal is compared any or multiple variation that presents in the following phenotype with the wild type animal: serum level of glucose reduces, body weight increases, fatty percent is higher.

9. claim 6,7 or 8 purposes or method, wherein said transgenic nonhuman animal is a rodent, preferred mouse.

10.Gpr100 polypeptide is used for identifying treatment, prevention Gpr100 relevant disease, preferred obesity or its activator of diabetes or the purposes of its antagonist, described Gpr100 polypeptide comprises the amino acid sequence shown in SEQ ID NO.3 or the SEQ ID NO:5, or with its at least 90% same sequence.

11.Gpr100 polynucleotide are used for identifying treatment, prevention Gpr100 relevant disease, preferred obesity or its activator of diabetes or the purposes of its antagonist, described Gpr100 polynucleotide comprise the nucleotide sequence shown in SEQID No.1, SEQ ID No.2 or the SEQ ID NO:4, or with its at least 90% same sequence.

12. non-human animal or its part, preferred cell, tissue or organ are used for the treatment of, prevent in evaluation or alleviate the Gpr100 relevant disease, the purposes in the activator of the Gpr100 polypeptide of preferred diabetes or obesity or the method for antagonist.

13. activator or antagonist that method by aforementioned any one claim or purposes are identified are used for the treatment of, prevent or alleviate Gpr100 relevant disease, the purposes of preferred obesity or diabetes.

14. the method for the adjusting of glucose, fat metabolism or weight increase in the individuality being regulated and control by the activity of Gpr100 polypeptide in the regulation and control individuality, described Gpr100 polypeptide comprises the amino acid sequence shown in SEQ ID NO.3 or the SEQ ID NO:5, or with its at least 90% same sequence.

15. the method for claim 14 comprises activator or the antagonist of described individuality being used Gpr100.

16. treatment suffers from the method for the individuality of Gpr100 relevant disease, described method comprises the active or amount that improves or reduce Gpr100 polypeptide in the described individuality.

17. the method for claim 16, wherein said method comprise described individuality is used the activator of Gpr100 polypeptide, Gpr100 polypeptide or the antagonist of Gpr100.

18. the method for diagnosis Gpr100 relevant disease said method comprising the steps of: (a) detect suffer from or the doubtful animal that suffers from described disease in Gpr100 polypeptide expression level or expression pattern; (b) expression or the expression pattern with described expression or expression pattern and intact animal compares.

19. the method for diagnosis Gpr100 relevant disease, described method are included in the variation that detects biological parameter as claimed in claim 5 in the doubtful individuality of suffering from described disease.

20.Gpr100 relevant disease, the diagnostic kit of preferred obesity or diabetes susceptibility comprises any or multiple in following: Gpr100 polypeptide or its part; Antibody at the Gpr100 polypeptide; Maybe can encode their nucleic acid.

21. the method for aforementioned each claim, wherein the Gpr100 relevant disease is selected from down the disease of group: obesity, comprise the prevention of obesity or weight increase, appetite suppresses, metabolism disorder, diabetes, comprise type i diabetes and type ii diabetes, and the associated conditions illness relevant with body weight, glucose tolerance is impaired, insulin resistance syndrome, the X syndrome, peripheral neurophaty, diabetic neuropathy, the albuminuria relevant with diabetes, the lipid metabolism illness comprises hyperglycemia, hyperlipidemia, dyslipidemia, hypertriglyceridemia, acute pancreatitis, angiocardiopathy, peripheral artery disease, hypertension, cardiomegaly, ischemic heart disease, hypercholesterolemia, obesity, prevention with obesity or weight increase.

22. reference and described hereinbefore basically method or purposes shown in Fig. 1 to 13 of accompanying drawing.

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