KR101524653B1 - A method for identifying the modulators of GPR43 - Google Patents

Abstract

The present invention relates to a method for evaluating the activity of a GPR43 (G protein receptor 43) regulator. Specifically, we developed a cell line capable of measuring cAMP with varying concentration depending on the activity of GPR43, developed a cell line that expresses GPR43 and GFP by fusion using GPR43 activation, and GPR43 is activated , A cell line expressing a fusion of a fragment of luciferase to GPR43 and beta-arrestin 2 is developed using the property of binding to beta-arrestin 2 and can be usefully used as an activity evaluation of GPR43 activity regulator .

Description

A method for identifying the modulators of GPR43

The present invention relates to a method for evaluating the activity of a GPR43 activity modulating substance.

G-protein coupled receptor (GPCR) is the largest cell membrane receptor, and approximately 800 GPCRs are found to be present in mammals. To date, GPCRs have been activated by a variety of extracellular stimuli such as neurotransmitters, hormones, cytokines, odorants, and light, Lt; / RTI > Among these, signal transduction materials such as specific proteins, peptides, amino acids, nucleic acids or fatty acid derivatives are secreted from specific cells to produce target cells having a specific receptor cell) to induce a cellular response. GPCR activates intracellular signal transduction through the activation of intracellular G-protein (Heterotrimeric G-protein). G-protein is activated by binding to GTP by receptor and then converted to GDP form, Is activated. Activated G-proteins then mediate down-signaling by inducing the activity of various signaling molecules in the cell (Effector). In vivo, GPCRs are known to play a pivotal role in mediating a variety of physiological or pathological phenomena as shown in the examples of chemokine receptors. Therefore, studies on GPCRs are now considered to be important targets for drug development as well as academic research subjects.

GPCRs are activated by the binding of extracellular ligands outside the cell to induce the activity of various intracellular G-proteins (Gq / 11, Gs, Gi / o, G12 / 13). The activated G-proteins may be selected from the group consisting of phospholipase C?, Adenylate cyclase, phosphoinositide-3-kinase, Ras and Rho family G- And induce the activity of the same signaling mediator molecule to transmit the receptor signal.

As its name suggests, GPCRs regulate the activity of G proteins (heterotrimeric G proteins) and deliver signals into cells. The G protein controls the α, β, and three divided into unit pieces, of which the Gα protein is a secondary bearer of Ca ^{2 +} and the concentration of the cells cAMP (second messenger) for γ. GPR43 raises the concentration of Ca ^{2 +,} then the concentration of cAMP is decreased when activated. Using this property, the method of measuring the concentration of Ca ^{2 +} with fluorescent dye was widely used. However, there was a disadvantage that the dye was expensive and the reaction time was short so that special equipment was used. In addition, CRE-luciferase and cAMP ELISA methods have been widely used as methods for measuring the concentration of cAMP, however, in the case of CRE-luciferase, measurement time is long and many parameters are disadvantageous. The cAMP ELISA was costly.

In order to overcome the disadvantages of the conventional methods, the present inventors have developed three methods for evaluating GPR43 activity using a glosensor system, cell image analysis, and protein-protein binding. Specifically, we developed a cell line capable of measuring cAMP with varying concentration depending on the activity of GPR43, developed a cell line that expresses GPR43 and GFP by fusion using GPR43 activation, and GPR43 is activated Using a property of binding to beta-arrestin 2, a cell line expressing a fusion of a fragment of luciferase to GPR43 and beta-arrestin 2, respectively, was developed and used for evaluation of activity of GPR43 activity regulator Thereby completing the present invention.

SUMMARY OF THE INVENTION [0006]

1) transforming a GPR43 (G protein receptor 43) gene into a Glosensor cell to produce a transformed cell;

2) culturing the transformed cells of step 1) in a reagent comprising luciferin;

3) treating and reacting the test substance in the cultured transformed cells of step 2); And

4) measuring the luminescence in the reacted transformed cells of step 3).

A further object of the present invention is to provide

1) transforming a vector containing GPR43 (G protein receptor 43) -GFP (green fluorescent protein) gene into a host cell to prepare a transformed cell;

2) treating the transformed cells of step 1) with a test substance and reacting;

3) staining nuclei of the transfected cells of step 2), and performing cell fluorescence image analysis to obtain a fluorescent spot image; And

4) quantifying the obtained fluorescent spot image of step 3) using a spot analysis program.

A further object of the present invention is to provide

1) preparing a vector expressing a fusion protein of firefly luciferase N-terminal domain and beta-arrestin2;

2) preparing a vector expressing a fusion protein of firefly luciferase C-terminal domain and GPR43 (G protein receptor 43);

3) co-transforming the vector of step 1) and the vector of step 2) into a host cell to produce transformed cells;

4) treating and reacting the test substance in the transformed cells of step 3); And

5) measuring the luminescence after addition of a sample containing luciferin to the reacted transformed cells of step 4).

In order to achieve the above object,

The present invention provides a method for preparing a transformed cell, comprising: 1) transforming a GPR43 (G protein receptor 43) gene into a Glosensor cell to produce a transformed cell;

2) culturing the transformed cells of step 1) in a reagent comprising luciferin;

3) treating and reacting the test substance in the cultured transformed cells of step 2); And

4) measuring the luminescence in the reacted transformed cells of step 3).

In order to achieve the above object,

The present invention provides a method for producing a transformed cell, comprising the steps of: 1) transforming a vector containing GPR43 (G protein receptor 43) -GFP (green fluorescent protein) gene into a host cell to prepare a transformed cell;

2) treating the transformed cells of step 1) with a test substance and reacting;

3) staining nuclei of the transfected cells of step 2), and performing cell fluorescence image analysis to obtain a fluorescent spot image; And

4) quantifying the obtained fluorescent spot image of step 3) using a spot analysis program.

In order to achieve the above object,

The present invention provides a method for producing a fusion protein comprising the steps of: 1) preparing a vector expressing a fusion protein of firefly luciferase N-terminal domain and beta-arrestin2;

2) preparing a vector expressing a fusion protein of firefly luciferase C-terminal domain and GPR43 (G protein receptor 43);

3) co-transforming the vector of step 1) and the vector of step 2) into a host cell to produce transformed cells;

4) treating and reacting the test substance in the transformed cells of step 3); And

5) measuring the luminescence after adding a sample containing luciferin to the reacted transformed cells of step 4); and evaluating the activity of the GPR43 activity modulating substance.

The present invention relates to a method for evaluating the activity of a GPR43 (G protein receptor 43) regulator. Specifically, we developed a cell line capable of measuring cAMP with varying concentration depending on the activity of GPR43, developed a cell line that expresses GPR43 and GFP by fusion using GPR43 activation, and GPR43 is activated , A cell line expressing a fusion of a fragment of luciferase to GPR43 and beta-arrestin 2 is developed using the property of binding to beta-arrestin 2 and can be usefully used for evaluating the activity of GPR43 activity regulator .

FIG. 1 is a diagram showing an overview of a glosensor system in which luciferase is activated when cAMP is bound.
Fig. 2 is a graph showing the activity of propionate in the gluosensor / hGPR43 cell line.
3 is a graph showing the activity evaluation by a substance known to modulate the activity of GPR43 in the gluosome / hGPR43 cell line.
FIG. 4 is a graph showing an excellent activity evaluation system by searching for a compound using the gluosome / hGPR43 cell line.
5 is a schematic diagram for explaining GPR43 modulating compounds using high-speed image equipment.
6 is a graph showing the results of quantification of the activity of GPR43 using cell image analysis using an in-apparatus analysis program (Spot Detector).
FIG. 7 is a graph showing a luciferase fragment complementation method for easily quantitatively measuring protein-protein binding in a cell.
FIG. 8 is a graph showing the results of quantitative analysis of binding of GPR43 and beta-arrestin 2 by an agonist of GPR43.

Hereinafter, the present invention will be described in detail.

The present invention

1) transforming a GPR43 (G protein receptor 43) gene into a Glosensor cell to produce a transformed cell;

2) culturing the transformed cells of step 1) in a reagent comprising luciferin;

3) treating and reacting the test substance in the cultured transformed cells of step 2); And

4) measuring the luminescence in the reacted transformed cells of step 3).

In this method, GPR43 of step 1) is preferably, but not necessarily, SEQ ID NO: 1.

In the method, the glow sensor cells of step 1) Glosensor ^TM cAMP HEK293 cells, but is not limited thereto.

In the above method, the reaction of step 2) is preferably carried out at 20 ° C for 2 hours, but is not limited thereto.

In the above method, the reaction of step 3) is preferably carried out at 20 ° C for 15 minutes, but is not limited thereto.

In this method, GPR43 is one of G-preotein-coupled receptor (GPCR), and G protein (heterotrimeric G protein) regulates the activity and delivers it to the cell. G protein is divided into three, alpha, beta and gamma. Among them, Gα protein regulates the intracellular concentration of Ca ^{2 +} and cAMP, which are secondary messengers. Therefore, when GPR43 is activated, Ca ²⁺ And decrease the intracellular concentration of cAMP.

The present invention

1) transforming a vector containing GPR43 (G protein receptor 43) -GFP (green fluorescent protein) gene into a host cell to prepare a transformed cell;

2) treating the transformed cells of step 1) with a test substance and reacting;

3) staining nuclei of the transfected cells of step 2), and performing cell fluorescence image analysis to obtain a fluorescent spot image; And

4) quantifying the obtained fluorescent spot image of step 3) using a spot analysis program.

In this method, GPR43 of step 1) is preferably, but not necessarily, SEQ ID NO: 1.

In this method, the vector of step 1) is preferably, but not limited to, pIRES-hGPR43-GFP-neo-puro.

In this method, the host cell of step 1) is preferably but not limited to a HeLa cell.

In this method, GPR43 is one of the GPCRs (G preotein-coupled receptors), and the GPCR receptor is bound to the agonist and then the GPCR receptor is transferred into the cells.

The present invention

1) preparing a vector expressing a fusion protein of firefly luciferase N-terminal domain and beta-arrestin2;

2) preparing a vector expressing a fusion protein of firefly luciferase C-terminal domain and GPR43 (G protein receptor 43);

3) co-transforming the vector of step 1) and the vector of step 2) into a host cell to produce transformed cells;

4) treating and reacting the test substance in the transformed cells of step 3); And

5) measuring the luminescence after adding a sample containing luciferin to the reacted transformed cells of step 4); and evaluating the activity of the GPR43 activity modulating substance.

In this method, the vector of step 1) is preferably but not limited to PB-CMV-MCS-EF1-Puro (SBI).

In this method, the firefly luciferase N-terminal domain of step 1) is preferably, but not limited to, SEQ ID NO: 2.

In this method, beta-arrestin 2 of step 1) is preferably SEQ ID NO: 3, but is not limited thereto.

In this method, the firefly luciferase C-terminal domain of step 1) is preferably, but not limited to, SEQ ID NO: 4.

In this method, the vector of step 2) is preferably but not limited to PB-EF1-MCS-IRES-neo (SBI)

In this method, GPR43 of step 2) is preferably, but not necessarily, SEQ ID NO: 1.

In this method, the host cell of step 3) is preferably, but not limited to, HEK 293 cells.

In the above method, when a firefly luciferase protein is separated into two fragments and β-arrestin 2 is fused to an N-terminal fragment and hGPR43 is fused to a C-terminal fragment, β-arrays A system was used in which the N-terminal and C-terminal fragments had the original enzyme activity due to the protein binding force between stin 2 and hGPR43.

In order to evaluate the activity of GPR43 using the cyclic AMP (cAMP) assay method of the present invention, Glosensor / hGPR43, in which GPR43 was stably expressed, was established and its activity was evaluated. As a result, The EC 50 of the following propionate was measured at 314 μM (see FIG. 2), and the synthetic compound represented by the following formula (2) was prepared as follows. EC50 was 160 nM, (EC 50 > 10 μM) and the synthetic compound (Chemical Formula 4) 066 had an EC50 of 416 nM (see FIG. 3). In addition, preliminary experiments were conducted using the Glosensor / hGPR43 cell line. As a result, the coefficient of variation (CV) of the Glosensor / hGPR43 cell line was 8.79% and the Z factor was 0.584, (See Fig. 4).

In order to evaluate the activity of GPR43 using the cell image analysis in the examples of the present invention, GPR43 was stably expressed by using the fact that the GPCR receptor migrates into the cell after binding with an agonist which is one of the characteristics of the GPCR receptor The GPR43-GFP was constructed and transfected into Hela cells. The fluorescence spot was treated with a high-speed fluorescence imaging device by treating the compound. As a result, fluorescence was observed in acetate, propionate, and 004 compounds compared with DMSO treatment Spot was formed (see Fig. 6A). In addition, it was confirmed that the negative evaluation group (065) did not form a spot, confirming that the activity evaluation system of the present invention was well formed (see FIG. 6B).

In order to evaluate the activity of GPR43 using protein-protein binding, Firefly luciferase protein was separated into two fragments, and β-arrestin 2 was fused to the N-terminal fragment and C-arrestin 2 to the C- When expressed in cells in the form of fusion of hGPR43, analysis was performed using a system in which N-terminal and C-terminal fragments had the original enzyme activity due to the protein binding force between? -areestin 2 and hGPR43 (see FIG. 7) As a result, it was confirmed that the treatment with the 004 compound was increased about 8-fold compared to the control group, as compared with about 2-fold to 3-fold increase in acetate and propionate (see FIG. 8).

Accordingly, a cell line capable of measuring the concentration-dependent cAMP according to the activity of GPR43 was developed as a method for evaluating the activity of the GPR43 (G protein receptor 43) regulatory substance of the present invention, and when the GPR43 was activated, And a cell line in which GPR43 and GFP are fused and expressed is developed. When GPR43 is activated, a cell line expressing a fusion of luciferase fragments to GPR43 and beta-arrestin, respectively, is obtained by using a property of binding to beta-arrestin2 And can be usefully used as an activity evaluation of GPR43 activity regulating substances.

Hereinafter, the present invention will be described in detail with reference to examples.

However, the following examples are illustrative of the present invention and are not intended to limit the scope of the present invention.

< Example  1> Cyclic AMP ( cyclic AMP , cAMP ) Measurement method GPR43 Activity evaluation

<1-1> GPR43 Establishment of stable animal cell line

A human GPR43-Myc gene was transfected into a Glosensor cell line to establish an animal cell line stably expressing GPR43. Specifically , the GPR43 gene was amplified from cDNA extracted from a human cell line using a PCR technique. Then, a glaucometer cell line (Promega) and a hGPR43-Myc gene (Stratagene) were inserted into a pIRES-neo3 vector. Fugene6 (Promega) The reagents were used to transduce pIRES-neo3-hGPR43-Myc. Single colony cells were separated and cultured in 500 .mu.g / ml DMEM (Welgene) supplemented with 10% fetal bovine serum (FBS, Invitrogen) and 200 .mu.g / ml hygromycin (Goldbio) and G418 And subcultured under the conditions of 5% CO 2 and 37 ° C. One cell line with relatively high activity among the established cell lines was selected and used in the subsequent experiments.

<1-2> Glow sensor / hGPR43  Activity evaluation of cell lines

Materials known to modulate the activity of GPR43 were evaluated. Specifically, the gluosensor / hGPR43 cell line of Example <1-1> was dispensed into a 96-well plate at 100,000 cells / well, and after 16-20 hours, the cells were suspended in a CO ₂ independent medium (Invitrogen) The cAMP reagent (Promega) containing luciferin was applied to the medium in a solution of 2% concentration. After 2 hours' reaction at 20 ° C, luminescence was measured in a combined instrument capable of measuring a luminometer or luminescence. At this time, the value is a value capable of correcting the cell number difference between the wells. GPR43 The propionate (Propionate, Sigma), the inventors of the present invention, synthesized the following [Formula 1] 004, [Formula 2] 065 and Formula 3] 066 according to the concentration gradient for 5 minutes Then, 1 μM Forskolin was treated for 15 minutes and reacted at 20 ° C. Then, the value obtained by measuring the luminescence again divided by the previous value is the final value, indicating the activity evaluation of GPR43.

As a result, the EC 50 of the natural ligand, propionate, was measured at 314 [mu] M (Fig. 2).

The synthetic compound, 004, was found to have an EC50 of 160 nM, a synthetic compound of 065, an EC 50 of 10 μM, and a synthetic compound of 066, of which the EC50 was 416 nM. These compounds were first reported (Lee et al., Identification and Functional Since the results were similar to those obtained with the Allosteric Agonists for the G Protein-Coupled Receptor FFA2, Mol. Pharmacol. 74: 1599 609, 2008, 004 EC50 = 480 nM, 066 EC50 = 700 nM, propionate EC50 = 125 μM) (Fig. 3).

<1-3> Glow sensor / hGPR43  Verification of compounds using cell lines

To search for compounds using the Glosensor / hGPR43 cell line, one plate (80 compounds) of a GPCR library composed of 5,000 compounds was randomly selected (Chembridge), 80 compounds were treated with 10 μM, (CV) values were obtained by calculating the mean and standard deviation of the values obtained by treating 80 compounds except for the control group, and then CV (%) = 100 X standard deviation / average.

As a result, the coefficient of variation (CV) of the Glosensor / hGPR43 cell line was 8.79% and the Z factor was 0.584. At this time, CV < 10% and Z factor > 0.5 are good criteria for the activity evaluation system. Therefore, the activity of CV was 8.79% and the Z factor was 0.584, which satisfied the criteria of good activity evaluation system (FIG. 4).

< Example  2> Using cell image analysis GPR43 Activity evaluation

<2-1> GPR43 - GFP Stable Animal cell line  Establish

One of the characteristics of the GPCR receptor is that it binds to an agonist and then moves the GPCR receptor into the cell. The present invention utilizes this property of GPCR to transduce HeLa cells as cervical cancer cells by binding green fluorescent protein (GFP) to a target protein hGPR43. Human GRP43 was cloned into pEGFP-N1 (Clontech) vector and then GFP was bound to human GRP43 by Clntech vector in order to clone the pIRESpuro3 (Clontech) vector in which a green fluorescent protein (GFP) And cloned into pIRESpuro3 (Clontech) vector using NheI and NotI. To transfect HeLa cells (ATCC) obtained from human cervical cancer tissue, HeLa cells were seeded at 1 × 10 ⁵ cells in a 12-well plate for 24 hours and then treated with lipofectamine 2000 (Invitrogen) Lt; / RTI &gt; 24 hours after transduction, single colonies surviving by treatment with 1 ug / mL puromycin (AGScientific) were obtained and stabilized by subculture. HeLa cells were cultured in DMEM (Hyclone, Dulbeco's Modified Eagle's Medium) supplemented with 10% fetal bovine serum, antibiotics, penicillin 100 units / ml + streptomycin 100 And incubated in an incubator with an environment of 5% CO 2, 95% air and 37 ° C temperature, and replaced with fresh medium from 2 days to 3 days, with addition of 0.5 ug / ml and pseudomonas (AGScientific) .

<2-2> GPR43 - GFP Quantification of Agents Expressed

HeLa cells (ATCC) stably expressing hGPR43-GFP were quantified using an analysis program (Spot Detector) in a high-speed fluorescence imaging apparatus after treatment of each compound. Specifically, the cells stabilized in Example <2-1> were divided into 8,000 cells / well in a 96-well plate and cultured for 16-18 hours. Then, 1 mM acetate (Sigma) 1, a natural ligand of hGPR43 mM propionate (Sigma) and 10 μM 004 compound for 30 minutes, fixed with 4% formalin at room temperature for 5 minutes, and rinsed with sterile water. 100 ng / mL Hoechst dye diluted in PBS was added and the nuclei were stained at room temperature for 5 minutes and washed three times with PBS. PBS was then added in 100 μL increments and the increased fluorescence spots were measured with a high-speed fluorescence imaging device (Cellomics ArrayScan, Thermo) (FIG. 5).

As a result, it was confirmed that fluorescence spots were formed in acetate, propionate and 004 compound, respectively, as compared with DMSO treatment (FIG. 6A). In addition, it was confirmed that the negative evaluation group (065) did not form a spot, confirming that the activity evaluation system of the present invention was well formed (FIG. 6B).

< Example  3> protein-protein binding GPR43 Activity evaluation

&Lt; 3-1 > Luciferase -β- Arrestin 2  And GPR43 -end Luciferase  Establishment of stable animal cell lines expressing each

Plasmids expressing N-terminal luciferase-beta-arrestin 2 and GPR43-C terminal luciferase were transfected into human embryonic kidney 293 (HEK293, ATCC) cell lines using Fugene6 (Promega) Lt; / RTI &gt; The transfected cells were treated with 1 μg / mL puromycin (AG Scientific) and 500 μg / mL G418 to obtain surviving single colonies, which were stabilized by subculture.

<3-2> Protein-protein binding GPR43 Activity evaluation

When Firefly luciferase protein is separated into two, and β-arrestin 2 is fused to the N-terminal fragment and hGPR43 is fused to the C-terminal fragment, the expression in the cell of β-arrestin 2 and hGPR43 And the N-terminal and C-terminal fragments have the original enzyme activity by the protein binding force (Fig. 7).

The hGPR43 /? - arrestin 2 cell line was divided into 100,000 cells / well on a 96-well white plate and cultured for 16 to 20 hours. Then, 1 mM acetate, 1 mM propionate and 10 μM 004 compound were treated for 30 minutes. ONE-Glo ^TM Luciferase 50 μL of the substrate-containing buffer solution in the ONE-Glo Luciferase Assay System (Promega) was added, and the activity was measured using a Luminometer (Berthold).

As a result, the treatment with the compound 004 increased about 8 times as compared with the control, while the acetate and propionate increased about 2 to 3 times (FIG. 8).

<110> Korea Research Institute of Bioscience and Biotechnology <120> A method for identifying the modulators of GPR43 <130> 12p-04-56 <160> 4 <170> Kopatentin 2.0 <210> 1 <211> 1709 <212> DNA <213> Homo sapiens <400> 1 gcactaggtc tggagagaca gcaaggtgct gtgcggcaga gcatttgggg tctcaaagaa 60 gcagtggcca ccaccatgga tacaggcccc gaccagtcct acttctccgg caatcactgg 120 ttcgtcttct cggtgtacct tctcactttc ctggtggggc tccccctcaa cctgctggcc 180 ctggtggtct tcgtgggcaa gctgcagcgc cgcccggtgg ccgtggacgt gctcctgctc 240 aacctgaccg cctcggacct gctcctgctg ctgttcctgc ctttccgcat ggtggaggca 300 gccaatggca tgcactggcc cctgcccttc atcctctgcc cactctctgg attcatcttc 360 ttcaccacca tctatctcac cgccctcttc ctggcagctg tgagcattga acgcttcctg 420 agtgtggccc acccactgtg gtacaagacc cggccgaggc tggggcaggc aggtctggtg 480 agtgtggcct gctggctgtt ggcctctgct cactgcagcg tggtctacgt catagaattc 540 tcaggggaca tctcccacag ccagggcacc aatgggacct gctacctgga gttccggaag 600 gaccagctag ccatcctcct gcccgtgcgg ctggagatgg ctgtggtcct ctttgtggtc 660 ccgctgatca 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ttcctctggg 600 tctactgggt tacctaaggg tgtggccctt ccgcatagaa ctgcctgcgt cagattctcg 660 catgccagag atcctatttt tggcaatcaa atcattccgg atactgcgat tttaagtgtt 720 gttccattcc atcacggttt tggaatgttt actacactcg gatatttgat atgtggattt 780 cgagtcgtct taatgtatag atttgaagaa gagctgtttt tacgatccct tcaggattac 840 aaaattcaaa gtgcgttgct agtaccaacc ctattttcat tcttcgccaa aagcactctg 900 attgacaaat acgatttatc taatttacac gaaattgctt ctgggggcgc acctctttcg 960 aaagaagtcg gggaagcggt tgcaaaacgc ttccatcttc cagggatacg acaaggatat 1020 gggctcactg agactacatc agctattctg attacacccg agggggatga taaaccgggc 1080 gcggtcggta aagttgttcc attttttgaa gcgaaggttg tggatctgga taccgggaaa 1140 acgctgggcg ttaatcagag aggcgaatta tgtgtcagag gacctatgat tatgtccggt 1200 tatgtaaaca atccggaagc gaccaacgcc ttgattgaca aggatggatg gctacattct 1260 ggagacatag cttactggga cgaagacgaa cacttcttca tagttgaccg cttgaagtct 1320 ttaattaaat acaaaggata tcaggtggcc cccgctgaat tggaatcgat attgttacaa 1380 caccccaaca tcttcgacgc gggcgtggca ggtcttcccg acgatgacgc cggtgaactt 1440 cccgccgccg ttgttgtttt ggagcacgga aagacgatg 1479 <210> 3 <211> 1735 <212> DNA <213> beta-arrestin2 <400> 3 ggtgagaagg ctgcgagcga gccgcgaacc cggcgggtgg agggcggcga gcgcgcgcgc 60 accatgggtg aaaaacccgg gaccagggtc ttcaagaagt cgagccctaa ctgcaagctc 120 accgtgtact tgggcaagcg tgactttgtg gatcacttgg acaaagtgga tcctgtcgat 180 ggtgtggtgc ttgtggatcc tgactacttg aaggaccgga aagtgtttgt gaccctcacc 240 tgtgccttcc gctatggccg agaagacctg gatgtactgg gcctgtcttt ccgcaaagat 300 ctgttcatcg ccacctacca ggccttcccc cccatgccca acccacctcg gccccccacc 360 cgcctacagg accgactgct gaagaagttg ggccagcatg cccacccctt ttttttcaca 420 ataccccaga atttgccttg ctccgtcaca ctgcagccag gaccggagga cacagggaag 480 gcctgtggag tagactttga gattcgagcc ttctgtgcca aatctataga agaaaaaagc 540 cacaaaagga actccgtgcg gcttatcatc agaaaggtac agtttgctcc tgagacaccc 600 ggcccccagc catcagctga aaccacacgc cacttcctca tgtctgaccg gaggtccctg 660 cacctagagg cttccctgga caaagagctg tactaccatg gggaacccct caatgtcaac 720 gtccacgtca ccaacaattc tgccaagacc gtcaagaaga tcagagtgtc tgtgagacag 780 tatgccgaca tttgcctctt cagcaccgcg cagtacaagt gtcctgtggc tcagcttgaa 840 caagatgacc aggtgtctcc cagttccaca ttctgcaagg tgtacaccat aaccccgctg 900 ctcagtgaca accgagagaa gcgtggcctt gcccttgatg ggcaactcaa gcacgaagac 960 accaacctgg cttccagcac cattgtgaag gagggagcca acaaggaggt gctgggaatc 1020 ctagtatcct acagggtcaa ggtgaagctg gtggtgtctc gaggcgggga tgtctccgtg 1080 gagctacctt tcgtcctaat gcaccccaag ccccacgacc acatcaccct tccccgaccc 1140 cagtcagccc cccgggaaat agacatccct gtggatacca acctcattga attcgatacc 1200 aactatgcca cagacgacga catcgtgttt gaggactttg cgaggcttcg gctgaagggg 1260 atgaaggatg acgactgtga tgaccagttc tgctaggaag aggggctggg agaggtaggg 1320 gtgggcagga ctgaggtccc actgcccttg cgggtaggag ggtcccagcc tctcctcctt 1380 ccccgtccgt ccacccgaga tacacactgg acccatcact cgttgaaagt gggcattaat 1440 cttttgactt cagctctgcc accccagccc tgctccctag ggtggcaagc tgtgtacaca 1500 cctaaagttt tgggaaggga acactgaaag caaggagtga atgtagagaa aaggagtaga 1560 aaggacaccc atgtttggcc tcataccaac ccactcccag tcaccacttc tgcctcccaa 1620 tccttgaaga tgagatttca ggggagggac cagcaaggcc atatcttcgt ttctgagcat 1680 agggaagaaa taaacctttt aataggcaaa aaaaaaaaaa aaaaaaaaaaaaaaa 1735 <210> 4 <211> 177 <212> DNA <213> Firefly luciferase C-terminus domain <400> 4 atgacggaaa aagagatcgt ggattacgtc gccagtcaag taacaaccgc gaaaaagttg 60 cgcggaggag ttgtgtttgt ggacgaagta ccgaaaggtc ttaccggaaa actcgacgca 120 agaaaaatca gagagatcct cataaaggcc aagaagggcg gaaagtccaa attgtaa 177

Claims (16)

1) transforming a GPR43 (G protein receptor 43) gene of SEQ ID NO: 1 into a Glosensor cell to produce a transformed cell;
2) culturing the transformed cells of step 1) in a reagent comprising luciferin;
3) treating and reacting the test substance in the cultured transformed cells of step 2); And
4) measuring the luminescence in the reacted transformed cells of step 3).


delete The method of claim 1 wherein the glow sensor cells of step 1) is Glosensor ^TM cAMP &lt; / RTI &gt; HEK293 cells.
The method according to claim 1, wherein the gluing agent cell of step 1) is measured in a well plate.
The method according to claim 1, wherein the reaction of step 2) is carried out at 20 ° C for 2 hours.
The method according to claim 1, wherein the reaction of step 3) is carried out at 20 ° C for 15 minutes to 2 hours.









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