KR101822932B1 - 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 for evaluating the activity 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 substance regulating GPR43 activity.

G-protein coupled receptor (GPCR) is a cell membrane receptor that occupies the largest part, and it has been found that about 800 GPCRs exist in mammals (Mammal). To date, GPCRs have been activated by various extracellular stimuli such as neurotransmitter, endocrine hormone, cytokine, odorants, and light, resulting in various cellular responses. Mediate. Among them, signaling substances such as specific proteins, peptides, amino acids, nucleic acids, or fatty acid derivatives are secreted from specific cells and target cells with specific receptors. cell) induces a cellular response. GPCR triggers intracellular signaling processes through the activity of intracellular G-proteins.G-proteins are activated by induction of binding to GTP by receptors, and are converted back to the form of GDP. It becomes active. Subsequently, activated G-proteins mediate sub-signaling by inducing the activity of various signaling molecules (Effectors) in the cell. In vivo, GPCRs are known to play a pivotal role in mediating various physiological or pathological phenomena, as shown in the example of chemokine receptors. Therefore, research on GPCR is considered not only as an academic research subject, but also as an important target for new drug development.

GPCRs are activated by the binding of extracelluar ligands outside the cell, inducing the activity of various intracellular G-proteins (Gq/11, Gs, Gi/o, G12/13). Activated G-proteins include phospholipase Cβ, adenylate cyclase, phosphoinositide-3-kinase, Ras and Rho family G-proteins. Receptor signals are transmitted by inducing the activity of the same sub-signaling mediator molecule.

GPCR, as its name suggests, transmits signals into cells by regulating the activity of heterotrimeric G proteins. The G protein is divided into three units of α, β and γ, of which the Gα protein regulates the intracellular concentration ^{of Ca 2 + and cAMP, which are secondary messengers.} When GPR43 is activated, ^{the concentration of Ca 2 +} increases and the concentration of cAMP decreases. Using this property, conventionally, ^{a method of measuring the concentration of Ca 2 +} with a fluorescent dye has been widely used. However, there was a disadvantage that the hair dye was expensive and the reaction time was short, requiring the use of special equipment. In addition, CRE-luciferase and cAMP ELISA have been widely used as a method of measuring the change in the concentration of cAMP, but CRE-luciferase has a disadvantage in that it takes a long time to measure and many variables are generated, The cAMP ELISA was expensive.

Accordingly, the present inventors have developed three methods for evaluating GPR43 activity using a Glosensor system, cell image analysis, and protein-protein binding in order to overcome the shortcomings of the existing methods. Specifically, a cell line capable of measuring cAMP whose concentration varies depending on the activity of GPR43 was developed, and a cell line in which GPR43 and GFP are fused and expressed using the property of moving into cells when GPR43 is activated was developed, and GPR43 is activated. Then, using the property of binding to beta-arrestin 2, a cell line that fused and expresses fragments of luciferase with GPR43 and beta-arrestin 2, respectively, can be developed and used to evaluate the activity of substances that regulate GPR43 activity. By revealing, the present invention was completed.

The object of the present invention is

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

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

3) reacting by treating the test material in the cultured transformed cells of step 2); And

4) It is to provide a method for evaluating the activity of a substance regulating GPR43 activity, comprising the step of measuring luminescence in the reacted transformed cells of step 3).

Another object of the present invention,

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

2) reacting the transformed cells of step 1) by treating the test material;

3) staining the nuclei of the transformed cells reacted in step 2), and then performing cell fluorescence image analysis to obtain a fluorescence spot image; And

4) It is to provide a method for evaluating the activity of a substance regulating GPR43 activity comprising the step of quantifying the fluorescence spot image obtained in step 3) using a spot analysis program.

Another object of 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) preparing a transformed cell by co-transforming the vector of step 1) and the vector of step 2) into a host cell;

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

5) It is to provide a method for evaluating the activity of a GPR43 activity modulating substance comprising the step of measuring luminescence after adding a sample containing luciferin to the reacted transformed cells in step 4).

To achieve the above object,

The present invention comprises the steps of 1) transforming a GPR43 (G protein receptor 43) gene into a Glosensor cell to prepare a transformed cell;

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

3) reacting by treating the test material in the cultured transformed cells of step 2); And

4) It provides a method for evaluating the activity of a substance regulating GPR43 activity, comprising the step of measuring luminescence in the reacted transformed cells of step 3).

To achieve the above object,

The present invention relates to 1) preparing a transformed cell by transforming a vector containing a GPR43 (G protein receptor 43)-GFP (green fluorescent protein) gene into a host cell;

2) reacting the transformed cells of step 1) by treating the test material;

3) staining the nuclei of the transformed cells reacted in step 2), and then performing cell fluorescence image analysis to obtain a fluorescence spot image; And

4) It provides a method for evaluating the activity of a substance regulating GPR43 activity comprising the step of quantifying the fluorescence spot image obtained in step 3) using a spot analysis program.

To achieve the above object,

The present invention comprises 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) preparing a transformed cell by co-transforming the vector of step 1) and the vector of step 2) into a host cell;

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

5) It provides a method for evaluating the activity of a substance regulating GPR43 activity comprising the step of measuring luminescence after adding a sample containing luciferin to the reacted transformed cells in step 4).

The present invention relates to a method for evaluating the activity of a substance regulating GPR43 (G protein receptor 43) activity. Specifically, a cell line capable of measuring cAMP whose concentration varies depending on the activity of GPR43 was developed, and a cell line in which GPR43 and GFP are fused and expressed using the property of moving into cells when GPR43 is activated was developed, and GPR43 is activated. Then, by using the property of binding to beta-arrestin 2, a cell line expressing by fusion of a luciferase fragment to GPR43 and beta-arrestin 2, respectively, can be developed and used to evaluate the activity of substances that regulate GPR43 activity. .

1 is a diagram showing an overview of a Glosensor system in which luciferase is activated when cAMP is bound.
2 is a graph showing the activity evaluation by propionate of the glosensor/hGPR43 cell line.
3 is a graph showing the evaluation of activity by a substance known to regulate the activity of GPR43 in the Glosensor/hGPR43 cell line.
4 is a graph confirming an excellent activity evaluation system by searching for a compound using the Glosensor/hGPR43 cell line.
5 is a diagram showing an outline for discovering a GPR43 modulating compound using an ultra-high-speed imaging device.
6 is a diagram showing the result of quantifying the activity of GPR43 using cell image analysis using an in-device analysis program (Spot Detector).
7 is a diagram showing a luciferase fragment complementation method for easily and quantitatively measuring protein-protein binding in cells.
8 is a diagram showing the result of quantifying and confirming the binding of GPR43 and beta-arrestin 2 by the agonist of GPR43.

Hereinafter, the present invention will be described in detail.

The present invention

1) preparing a transformed cell by transforming the GPR43 (G protein receptor 43) gene into a Glosensor cell;

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

3) reacting by treating the test material in the cultured transformed cells of step 2); And

4) It provides a method for evaluating the activity of a substance regulating GPR43 activity, comprising the step of measuring luminescence in the reacted transformed cells of step 3).

In the above method, GPR43 in step 1) is preferably SEQ ID NO: 1, but is not limited thereto.

In the above method, the glosensor cell of step 1) ^{is preferably a Glosensor TM} cAMP HEK293 cell, but is not limited thereto.

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

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

In the above method, GPR43 is one of G preotein-coupled receptors (GPCR) and heterotrimeric G protein (G protein) regulates its activity and delivers it into cells. G protein is divided into three units of α, β, and γ, of which Gα protein ^{regulates the intracellular concentrations of the secondary messengers Ca 2+} and cAMP, so when GPR43 is also active, the concentration of ^{Ca 2+ is controlled.} The principle of increasing and decreasing the intracellular concentration of cAMP was used.

The present invention

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

2) reacting the transformed cells of step 1) by treating the test material;

3) staining the nuclei of the transformed cells reacted in step 2), and then performing cell fluorescence image analysis to obtain a fluorescence spot image; And

4) It provides a method for evaluating the activity of a substance regulating GPR43 activity comprising the step of quantifying the fluorescence spot image obtained in step 3) using a spot analysis program.

In the above method, GPR43 in step 1) is preferably SEQ ID NO: 1, but is not limited thereto.

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

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

In the above method, GPR43 is one of the GPCRs (G preotein-coupled receptors), and the GPCR receptor is bound to an agonist and then the GPCR receptor moves into the cell.

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) preparing a transformed cell by co-transforming the vector of step 1) and the vector of step 2) into a host cell;

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

5) It provides a method for evaluating the activity of a substance regulating GPR43 activity comprising the step of measuring luminescence after adding a sample containing luciferin to the reacted transformed cells in step 4).

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

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

In the above method, beta-arrestin2 in step 1) is preferably SEQ ID NO: 3, but is not limited thereto.

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

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

In the above method, GPR43 in step 2) is preferably SEQ ID NO: 1, but is not limited thereto.

In the above method, the host cell of step 3) is preferably a HEK 293 cell, but is not limited thereto.

In the above method, when the firefly luciferase protein is separated into two, β-arrestin2 is fused to the N-terminal fragment and hGPR43 is fused to the C-terminal fragment, and expressed in cells. A system in which the N-terminal and C-terminal fragments have the original enzymatic activity by the protein binding force between Stine2 and hGPR43 was used.

In an embodiment of the present invention, in order to find out the activity evaluation of GPR43 using the cyclic AMP (cAMP) measurement method of the present invention, a GPR43 stably expressed Glosensor/hGPR43 was established and the activity was evaluated. The EC 50 of the following [Formula 1] propionate was measured to be 314 μM (see FIG. 2), and the following [Formula 2] 004, which is a synthetic compound, has an EC50 of 160 nM, and the following [Formula 3] 065 EC 50> 10 μM and the following [Chemical Formula 4] 066, which is a synthetic compound, has an EC50 of 416 nM, which proved to be a good activity evaluation system (see FIG. 3). In addition, as a result of conducting a preliminary experiment for compound search using the Glosensor/hGPR43 cell line, the coefficient of variation (CV) of the Glosensor/hGPR43 cell line was 8.79% and the Z factor was 0.584, indicating excellent activity. The criteria of the activity evaluation system were satisfied (see FIG. 4).

In an embodiment of the present invention, in order to evaluate the activity of GPR43 using cell image analysis, GPR43 is stably expressed by using the fact that the GPCR receptor moves into the cell after binding with an agonist, which is one of the characteristics of the GPCR receptor. GPR43-GFP was established and transduced into Hela cells, and as a result of processing the compound and confirming the fluorescent spot with an ultra-high-speed fluorescence imaging equipment, the result of fluorescing in acetate, propionate, and 004 compounds, respectively, compared to DMSO-treated compounds. It was confirmed that a spot was formed (see FIG. 6A). In addition, it was confirmed that the 065 compound, which is a negative control group, did not form spots, and that the activity evaluation system of the present invention was well made (see FIG. 6B).

In an embodiment of the present invention, in order to evaluate the activity of GPR43 using protein-protein binding, a firefly luciferase protein was separated into two, and β-arrestin2 was fused to the N-terminal fragment, and the C-terminal fragment was When expressed in cells in the form of fusion of hGPR43, the N-terminal and C-terminal fragments have the original enzymatic activity due to the protein binding force between β-arrestin2 and hGPR43. As a result, it was confirmed that the amount of acetate and propionate increased by about 2 to 3 times, whereas the treatment with the 004 compound increased by about 8 times compared to the control (see FIG. 8).

Therefore, as a method for evaluating the activity of a substance regulating GPR43 (G protein receptor 43) activity of the present invention, a cell line capable of measuring cAMP whose concentration varies according to the activity of GPR43 was developed. Thus, a cell line was developed in which GPR43 and GFP were fused and expressed, and when GPR43 was activated, a cell line that expressed by fusion of a luciferase fragment to GPR43 and beta-arrestin, respectively, was developed using the property of binding to beta-arrestin 2. It can be developed and usefully used as an activity evaluation of a substance that modulates GPR43 activity.

Hereinafter, the present invention will be described in detail by examples.

However, the following examples specifically illustrate the present invention, and the contents of the present invention are not limited by the examples.

< Example 1> Cyclic AMP ( cyclic AMP , cAMP ) evaluation method of activity of GPR43 using measurement method

<1-1> Establishment of animal cell lines stably expressing GPR43

In order to establish an animal cell line stably expressing GPR43, a human GPR43-Myc gene was transduced into a Glosensor cell line. Specifically , the GPR43 gene was amplified from cDNA extracted from a human cell line using a PCR technique, and then the glosensor cell line (Promega) and the hGPR43-Myc gene (Stratagene) were inserted into the pIRES-neo3 vector, and Fugene6 (Promega) into the glosensor cell line. The reagent was used to transduce pIRES-neo3-hGPR43-Myc. Medium in which 10% fetal bovine serum (Fetal Bovine Serum, FBS, Invitrogen) and 200 μg/ml hygromycin (Goldbio) and G418 (Goldbio) were added to 500 μg/ml DMEM (Welgene) by separating single colony cells Was subcultured under conditions of 5% CO2 and 37°C. Among the established cell lines, one cell line having relatively excellent activity was selected and used in subsequent experiments.

<1-2> Glosensor/hGPR43 cell line activity evaluation assay

Substances known to modulate the activity of GPR43 were evaluated. Specifically, the cell line of Example <1-1> Glosensor/hGPR43 cells were dispensed into a 96 well plate at 100,000 cells/well, and 16-20 hours later, in CO ₂ independent media (Invitrogen). A solution in which a cAMP reagent (Promega) containing luciferin was mixed at a concentration of 2% was spread on the medium. After reaction at 20° C. for 2 hours, luminescence was measured in a luminometer or a complex device capable of measuring luminescence. In this case, the value is a value capable of correcting the difference in the number of cells between wells. Compounds that modulate GPR43 [Formula 1] Propionate (Sigma), the inventor? The following [Formula 1] 004, [Formula 2] 065 and [Formula 3] 066 were treated for 5 minutes according to the concentration gradient, and then 1 μM Forskolin was treated for 15 minutes and reacted at 20°C. Then, the value obtained by measuring the luminescence again and dividing the result by the previous value was the final value, indicating the evaluation of the activity of GPR43.

As a result, the EC 50 of the natural ligand propionate was measured to be 314 μM (FIG. 2).

In addition, the synthetic compound 004 has an EC50 of 160 nM, the synthetic compound 065 has an EC 50> 10 μM, and the synthetic compound 066 has an EC50 of 416 nM (Lee et al., Identification and Functionality). Characterization of Allosteric Agonists for the G Protein-Coupled Receptor FFA2, Mol. Pharmacol. 74:1599 609, 2008, 004 EC50=480nM, 066 EC50=700nM, propionate EC50=125μM). Proved to be a good activity evaluation system (Fig. 3).

<1-3> Compound verification using Glosensor/hGPR43 cell line

In order to search for compounds using the Glosensor/hGPR43 cell line, one plate (80 compounds) from the GPCR library (Library) consisting of 5000 compounds was randomly selected (Chembridge), and 80 compounds were treated with 10 μM, and the control and 004 compounds were used. The treated group was included in a 96-well plate, and the coefficient of variation (CV) was calculated by calculating the mean and standard deviation of the values obtained by treating 80 compounds excluding the control group, and then CV (%) = 100 Calculated by the formula of X standard deviation/mean.

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, when CV <10% and Z factor> 0.5, it is the standard of an excellent activity evaluation system. Therefore, the CV was 8.79% and the Z factor was 0.584, showing excellent activity, satisfying the criteria of a good activity evaluation system (Fig. 4).

<Example 2> Method for evaluating the activity of GPR43 using cell image analysis

<2-1> Establishment of stable animal cell line expressing GPR43-GFP

One of the characteristics of the GPCR receptor is that after binding to an agonist, the GPCR receptor moves into the cell. In the present invention, using this characteristic of GPCR, a green fluorescent protein (GFP) was bound to a target protein, hGPR43, and transduced into HeLa cells, which are cervical cancer cells. In order to clone a form in which green fluorescent protein (GFP) is bound to human GPR43 in pIRESpuro3 (Clontech) vector, human GRP43 is first cloned into pEGFP-N1 (Clontech) vector, and then GFP is bound to human GRP43 using NheI and NotI. It was cloned into pIRESpuro3 (Clontech) vector using NheI and NotI. In order to transduce the plasmid into HeLa (ATCC), a cell obtained from human cervical cancer tissue, 1 X 10 ⁵ cells were planted in each well in a 12-well plate, and then 24 hours later, lipofectamine 2000 (Invitrogen) was used. And transduced. 24 hours after transduction, 1 ug/mL puromycin (AGScientific) treatment was performed to obtain a single colony that survived, which was subcultured to stabilize. The growth medium of HeLa cells is DMEM (Hyclone, Dulbeco's Modified Eagle's Medium), 10% fetal bovine serum, antibiotics, penicillin 100 units/ml + streptomycin 100 Μg/ml and puromycin (AGScientific) 0.5 ug/mL were added, and cultured in an incubator having an environment of 5% CO2, 95% air, and 37°C temperature, and replaced with fresh medium on the 2nd to 3rd day. .

<2-2> Quantification of agent expressing GPR43-GFP

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

As a result, it was confirmed that fluorescent spots were formed in acetate, propionate, and compound 004, respectively, compared to those treated with DMSO (FIG. 6A). In addition, it was confirmed that the 065 compound, which is a negative control group, did not form spots, and that the activity evaluation system of the present invention was well made (FIG. 6B).

<Example 3> Method for evaluating the activity of GPR43 using protein-protein binding

<3-1> N-terminal Establishment of stable animal cell lines expressing luciferase- β- Arrestin2 and GPR43 -terminal luciferase, respectively

Two plasmids using Fugene6 (Promega) reagent in human embryonic kidney (Human Embryonic Kidney 293, HEK293, ATCC) cell lines expressing N-terminal luciferase-β-arrestin2 and GPR43-C-terminal luciferase. Was transduced. The transduced cells were treated with 1 μg/mL puromycin (puromycin, A. G. Scientific) and 500 μg/mL G418 to obtain a surviving single colony, which was subcultured to stabilize.

<3-2> Evaluation of GPR43 activity using protein-protein binding

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. It is a system in which the N-terminal and C-terminal fragments have the original enzymatic activity by protein binding force (Fig. 7).

hGPR43/β-Arrestin 2 cell line was dispensed into a 96-well white plate at 100,000 cells/well, cultured for 16 to 20 hours, and then treated with 1 mM acetate, 1 mM propionate, and 10 μM 004 compound for 30 minutes. ^{ONE-Glo TM} right into your cells Luciferase 50 μL of a buffer solution containing a substrate present in the assay method (ONE-Glo Luciferase Assay System, Promega) was added each, and the activity was measured using a measuring instrument (Luminometer, Berthold).

As a result, the amount of acetate and propionate increased by about 2 to 3 times, whereas the treatment with compound 004 increased by about 8 times compared to the control group (FIG. 8).

<110> Korea Research Institute of Bioscience and Biotechnology <120> A method for identifying the modulators of GPR43 <130> 2017P-04-033 <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 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gagtcaggag cgggggagac agggctccag ggatgaggcc gcattctgct 1560 cccacagcgc cttttccaga aagttcccat tgctcaataa atgtggatca tcagagacat 1620 ttatgaacaa tgacagaaga aaaattaccc aaataaatgt ggaagcaagc aaaaaaaaaa 1680 aaaaaaaaaa aaaaaaaaaa aaaaaaaaa 1709 <210> 2 <211> 1479 <212> DNA <213> Firefly luciferase) N-terminus domain <400> 2 atggaagacg ccaaaaacat aaagaaaggc ccggcgccat tctatcctct agaggatgga 60 accgctggag agcaactgca taaggctatg aagagatacg ccctggttcc tggaacaatt 120 gcttttacag atgcacatat cgaggtgaac atcacgtacg cggaatactt cgaaatgtcc 180 gttcggttgg cagaagctat gaaacgatat gggctgaata caaatcacag aatcgtcgta 240 tgcagtgaaa actctcttca attctttatg ccggtgttgg gcgcgttatt tatcggagtt 300 gcagttgcgc ccgcgaacga catttataat gaacgtgaat tgctcaacag tatgaacatt 360 tcgcagccta ccgtagtgtt tgtttccaaa aaggggttgc aaaaaatttt gaacgtgcaa 420 aaaaaattac caataatcca gaaaattatt atcatggatt ctaaaacgga ttaccaggga 480 tttcagtcga tgtacacgtt cgtcacgtct catctacctc ccggttttaa tgaatacgat 540 tttgtaccag agtcctttga tcgtgacaaa acaattgcac tgataatgaa 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 aaaaaaaaaa aaaaa 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 (3)

1) transforming a host cell with a vector comprising a fusion gene of GPR43 (G protein receptor 43) and GFP (green fluorescent protein), which is composed of SEQ ID NO: 1, to produce transformed cells;
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.
2. The method according to claim 1, wherein the vector of step 1) is plRES-hGPR43-GFP-neo-puro.
The method according to claim 1, wherein the host cell of step 1) is a HeLa cell.

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