CN103966171A - Cell line for screening peptide and non-peptide GLP-1 (Glucagon-Like Peptide 1) analogs as well as preparation method and application of cell line - Google Patents
Cell line for screening peptide and non-peptide GLP-1 (Glucagon-Like Peptide 1) analogs as well as preparation method and application of cell line Download PDFInfo
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- CN103966171A CN103966171A CN201410234121.2A CN201410234121A CN103966171A CN 103966171 A CN103966171 A CN 103966171A CN 201410234121 A CN201410234121 A CN 201410234121A CN 103966171 A CN103966171 A CN 103966171A
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- glp
- peptide
- cell
- u2os
- u2os cell
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- DTHNMHAUYICORS-KTKZVXAJSA-N Glucagon-like peptide 1 Chemical class C([C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCCN)C(=O)NCC(=O)N[C@@H](CCCNC(N)=N)C(N)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@H](CCC(N)=O)NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CC=1N=CNC=1)[C@@H](C)O)[C@@H](C)O)C(C)C)C1=CC=CC=C1 DTHNMHAUYICORS-KTKZVXAJSA-N 0.000 title claims abstract description 37
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0693—Tumour cells; Cancer cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2503/00—Use of cells in diagnostics
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2510/00—Genetically modified cells
Abstract
The invention relates to a cell line for screening peptide and non-peptide GLP-1 analogs and an application of the cell line and belongs to the technical field of high-flux medicine screening and detection. The invention relates to an establishment of a cell line GLP-1R / U2OS for stably expressing fluorescently-labeled human glucagon-like peptide 1 receptor (GLP-1R) protein and a method for detecting the biological activity of GLP-1 analogs by observing and analyzing morphological changes via a high-content system on the basis that fluorescent spots are formed in cells after the GLP-1R/U2OS cell line is stimulated by Exendin-4 as a GLP-1 analog. The detection method is easily standardized and has the characteristics of good repeatability, GLP-1 receptor specificity, low cost, accuracy and convenience, and has good application prospects.
Description
Technical field:
The invention belongs to medicament high flux screening detection technique field, particularly, relate to a kind of fluorescent mark human glucagon-like-peptide-1 acceptor (GLP-1R) albuminous cell strain GLP-1R/U2OS cell strain and establishment method thereof of expressing, and the metamorphosis that produces fluorescence spot after being subject to glucagon-like peptide-1 analogs and stimulating based on this cell strain in cell is measured the detection method of glucagon-like peptide-1 analogs biological activity and receptor stimulant, antagonist and conditioning agent.
Background technology:
Incretin is to increase the hormone that gi tract discharge when insulin secretion in nutrition absorption.Two kinds of enteron aisle peptides that account for the most of effects of incretin are: 1) GLP-1 (Glucagon-like peptide, glucagon-like peptide 1): be 30 or 31 the amino acid whose hormones that contain of being secreted by small intestine L cell (Langerhans cell) by Proglucagon genes encoding, under nutritive substance stimulates, GLP-1 brings into play the secretion of promotion Regular Insulin and the effect of glucagon suppression secretion by acting on pancreatic beta cell, and GLP-1 and acceptor thereof are distributed widely in central nervous system, pancreas and gastrointestinal system.2) GIP (Gastric inhibition polypeptide orglucose-dependent insulinotropic peptide, glucose-dependent-insulinotropic polypeptide): by duodenum and near-end jejunum K emiocytosis.In the several minutes discharging from small intestine position, GLP-1 is carried out rapid metabolism (proteolytic cleavage) with GIP by dipeptidyl peptidase-IV (DPP-IV) and becomes non-activity metabolite.The acceptor site that active hormones arrival pancreas acts on beta-cell in a small amount stimulates insulin secretion in dependence on the glucose mode; And GLP-1 acts on the secretion of alpha cell and glucagon suppression.It is that DPP-IV inhibitor (as Xi Gelieting, BI 1356 etc.) and GLP-1 receptor stimulant (are treated based on Exendin-4, as Exenatide that treatment based on incretin at present is mainly divided into two large classes; People GLP-1 analogue, as Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37]).
GLP-1 is considered to most important incretin at present, and it is bringing into play the approximately incretin activity of 70-80%, and all these physiological characteristics all show that GLP-1 will play a significant role in the treatment of diabetes B.Novel ofhypoglycemic medicine GLP-1 receptor stimulant can reduce glycolated hemoglobin level significantly, lastingly, and has rapidly, efficiently and reduce blood sugar, improves islet beta cell function, reduces the effects such as body weight.No matter be animal level or cell levels, GLP-1 receptor stimulant all shows the physiological action of hyperglycemia; Promote the release of Regular Insulin, the secretion of glucagon suppression, postpones stomach emptying, promotes propagation and the regeneration of β cell, suppresses the apoptosis of β cell.
Although the correlative study of GLP-1 and analogue thereof has obtained good progress, multiple analogue has also entered clinical experimental stage, but because polypeptide drug has a common drawback, can not oral administration, can only be by subcutaneous injection, therefore for type-II diabetes (T2DM) patient of long-term prescription, its compliance is still not high.Due to the characteristic of polypeptide drug, its stability dependency is in time, temperature and pH value, and storage conditions is had relatively high expectations.In addition, because these agonists have all been introduced foreign protein to human body, may cause the immune response of body.The GLP-1 analogue of listing is expensive at present, can increase the weight of patient's economical load.Screening small molecules GLP-1 analogue has become study hotspot at present.
Setting up the bioactive measuring method of effective GLP-1 analogue, is one of key measure of evaluating new drug drug effect.
Summary of the invention:
The object of this invention is to provide a kind of peptide class of new in-vitro screening glucagon-like peptide-1 receptor and the method for non-peptide excitomotor, conditioning agent and antagonist, a kind of fluorescent mark human glucagon-like-peptide-1 acceptor (GLP-1R) albuminous cell strain GLP-1R/U2OS cell strain and establishment method thereof of expressing is provided, the C-terminal of the direct mark GLP-1R of XFP, and metamorphosis based on this cell strain is subject to producing in cell after glucagon-like peptide-1 analogs stimulates fluorescence spot is measured the bioactive detection method of glucagon-like peptide-1 analogs.
The present invention is achieved above-mentioned purpose of the present invention with following technical scheme:
One strain GLP-1R/U2OS cell strain, obtained by following method: build human glucagon-like-peptide-1 acceptor (GLP-1R) fluorescin (XFP) carrier for expression of eukaryon GLP-1R/pCMV6, and stable transfection human osteosarcoma U2OS cell, obtain a strain GLP-1R/U2OS cell strain.
The fluorescin (XFP) that described GLP-1R/U2OS cell strain contains is GFP (greenfluorescence protein), YFP (yelow fluorescence protein), RFP (red fluorescenceprotein) or any albumen with photoluminescent property.
Described GLP-1R/U2OS cell strain or other applicable cell are as HEK293 (humanembroynic kidney293) cell, obtained by following method: first build human glucagon-like-peptide-1 acceptor (GLP-1R) green fluorescent protein (GFP) carrier for expression of eukaryon GLP-1R/pCMV6, then carry out transfection U2OS cell by Lipofectamine2000 process specifications.G418 (400 μ g/ml) pressurization screening 2 weeks, obtains the positive cell clone with antibiotics resistance, and amplification cultivation is the U2OS clone of stably express people GLP-1R, called after GLP-1R/U2OS cell strain.
By the described bioactive method of GLP-1R/U2OS cell strain mensuration GLP-1 analogue, transfection under the condition of GLP-1R gene of XFP mark, under receptor stimulant stimulates, induce in GLP-1R/U2OS cell and produce fluorescence spot, the GLP-1R of transfection can stably express.
As described in method, wherein said receptor stimulant is GLP-1 analogue, the activity that produces fluorescence spot intensity and receptor stimulant after GLP-1 binding of receptor and ligand in cell is proportionate, analyze and measure fluorescence spot intensity in GLP-1R/U2OS cell with high intension systematic observation, measure the biological activity of receptor stimulant.
As described in method, wherein said GLP-1 analogue is insulin secretion accelerating peptide Exendin-4.
As described in method, by the expression result of high intension systematic observation GLP-1R, comprise GLP-1 receptor stimulant, antagonist and conditioning agent etc. with fluorescence spot average intensity change detection GLP-1 analogue in GLP-1R/U2OS cell and GLP-1R bio-active substance.
The invention provides the foundation of stably express fluorescent mark human glucagon-like-peptide-1 acceptor (GLP-1R) albuminous cell strain GLP-1R/U2OS, and be subject to forming fluorescence spot in the rear cell of GLP-1 analogue Exendin-4 stimulation based on GLP-1R/U2OS cell strain, analyze metamorphosis with high intension systematic observation and measure the bioactive detection method of GLP-1 analogue.The easy stdn of this detection method, reproducible, have that GLP-1 receptor-specific, cost are low, feature accurately and easily, have a good application prospect.
The method of current domestic screening GLP-1 receptor stimulant be by the gene of GLP-1 acceptor and reporter gene cotransfection in cell, and by GLP-1 expression of receptor at cell surface, by stimulating in rear cell cAMP level to change, agonist is screened.The invention provides fluorescent mark human glucagon-like-peptide-1 acceptor (GLP-1R) albuminous cell strain GLP-1R/U2OS, compare domestic screening method and there is quick, sensitive, specificity advantages of higher, and can greatly reduce false-positive generation, and can screen for mixture, also can reduce testing cost simultaneously.
Brief description of the drawings:
Fig. 1 XFP mark GLP-1R schematic diagram;
Fig. 2 recombinant eukaryon expression vector GLP-1R/pCMV6 schematic diagram;
The expression result of the high intension systematic observation of Fig. 3 analysis of cells surface GLP-1R;
The high intension systematic observation of Fig. 4 is analyzed to stimulate in rear cell and is formed fluorescence spot;
Fig. 5 GLP-1R/U2OS cell is active reaction curve result after Exendin-4 activates.
Embodiment:
Below in conjunction with accompanying drawing, further illustrate essentiality content of the present invention with embodiments of the invention, but do not limit the present invention with this.
Embodiment 1:
The structure of GFP fluorescent mark people GLP-1R carrier for expression of eukaryon GLP-1R/pCMV6, the foundation of GLP-1R/U2OS cell strain, and fluorescence spot average intensity change detection GLP-1 analogue biological activity in GLP-1R/U2OS cell.
One, the structure of GFP fluorescent mark people GLP-1R carrier for expression of eukaryon GLP-1R/pCMV6:
People GLP-1R cDNA amplification: taking people GLP-1R cDNA as template, adopt the mode of the PCR total length people GLP-1R cDNA coding region of increasing, total length 1392bp.
Its upstream primer: 5 '-gaggcgatcgccATGGCCGGCGCCCCCGGC-3 ';
Downstream primer: 5 '-gcgacgcgtGCTGCAGGAGGCCTGG CAAG-3 '.
2. recovery, purified pcr product, be connected on pMD18-T Vector, obtains recombinant plasmid GLP-1R/pMD18-T, utilizes restriction enzyme Sgf I and Mlu I to carry out enzyme to plasmid and cut.
3. with restriction enzyme Sgf I and Mlu I digested plasmid G-D (pCMV6-AC-GFP-DR5).
4. reclaim enzyme and cut product, connect 8 hours at 16 DEG C, obtain recombinant plasmid GLP-1R/pCMV6, go intracellular toxin plasmid to extract in a large number test kit specification sheets according to OMEGA and extract GLP-1R/pCMV6 plasmid.
The clone PCR reaction system of gene order is 50 μ L, is made up of 5 μ L10 × Ex-Taq buffer, 1 μ LEx-Taq archaeal dna polymerase 5U/ μ L, 3 μ L2.5mM dNTP, 1 μ L upstream primer 10pmol/ μ L, 1 μ L downstream primer 10pmol/ μ L, 1 μ L plasmid (BC113493) 100ng/ μ L and 38 μ L sterilized waters; PCR reaction conditions: 95 DEG C of denaturation 5min, 95 DEG C of sex change 1min, 65 DEG C of annealing 0.5min, 72 DEG C are extended 1min, totally 30 circulations, 72 DEG C are extended 10min; In 10 × Ex-Taq buffer used, contain Mg
2+.
It is 10 μ L that recovery PCR product is connected to pMD18-T Vector reaction system, is made up of 5 μ L recovery PCR products, 4.5 μ L SolutionI, 0.5 μ L pMD18-T Vector; Reaction conditions: connect 4 hours at 16 DEG C.
Double digestion plasmid GLP-1R/pMD18-T reaction system is 50 μ L, is made up of 2 μ L Sgf I (5U/ μ L), 2 μ L Mlu I (5U/ μ L), 5 μ L10 × Tango Buffer, 25 μ LGLP-1R/pMD18-T and 16 μ L sterilized waters; Reaction conditions: 37 DEG C of incubations 2 hours.
Double digestion plasmid G-D (pCMV6-AC-GFP-DR5) reaction system is 50 μ L, is made up of 2 μ L Sgf I (5U/ μ L), 2 μ L Mlu I (5U/ μ L), 5 μ L10 × Tango Buffer, 10 μ LG-D and 31 μ L sterilized waters; Reaction conditions: 37 DEG C of incubations 2 hours.
Construction recombination plasmid GLP-1R/pCMV6 is with the restriction enzyme digestion product in step (2) and (3), connects 8 hours under the condition of 16 DEG C; Reaction system is 11 μ L, by the plasmid pCMV6 fragment after restriction enzyme digestion in 5 μ L SolutionI, step (4) with GLP-1R gene order fragment is respectively 1 μ L and 5 μ L form; Reaction conditions: connect 8 hours at 16 DEG C.
The plasmid obtaining in step (4) further Transformed E .coli DH5 α is preserved amplification.
Two, the foundation of the U2OS clone of stable transfection people GLP-1R:
U2OS cell is containing in the DMEM substratum of 10% (volume fraction) calf serum, in 37 DEG C, 5% (volume fraction) CO
2saturated humidity under cultivate.Carry out the transfection of recombinant expression vector GLP-1R/pCMV6 by Lipofectamine2000 process specifications until cytogamy during to 70%-80%.After transfection 48h, with trypsin digestion and cell, pressurization screening 2 weeks in the selection substratum that contains G418 (400 μ g/ml), acquisition has the positive cell clone of antibiotics resistance, amplification cultivation is the U2OS clone of stably express people GLP-1R, called after GLP-1R/U2OS cell strain.By the expression result of high intension systematic observation GLP-1R.
Three, fluorescence spot average intensity change detection GLP-1 analogue biological activity in GLP-1R/U2OS cell:
The GLP-1R/U2OS cell of above-mentioned acquisition is incubated to 96 well culture plates by 100 μ L/ holes (1000 cells/well), with the DMEM substratum containing 10% (volume fraction) calf serum, 37 DEG C, 5%CO
2under condition, cultivate 24 hours.Within second day, remove perfect medium, use PBS buffer solution for cleaning, add different concns sample to be tested Exendin-4, and solution removed hole after 20 minutes in, fix 30 minutes by 4% paraformaldehyde room temperature, wash 3 times with PBS damping fluid, add DAPI dyestuff lucifuge dyeing 15 minutes, wash 5 times with PBS damping fluid, in hole, stay 100 μ LPBS damping fluids, with high intension systematic observation analysis of fluorescence spot, and calculate spot average intensity with Transfluor module analysis, make typical curve with Excel.
Embodiment 2:
The expression of GLP-1R on GLP-1R/U2OS cytolemma:
GLP-1R/U2OS cell is incubated at 96 well culture plates by 100 μ L/ holes (1000 cells/well), with the DMEM substratum containing 10% (volume fraction) calf serum, 37 DEG C, 5%CO
2under condition, cultivate 24 hours.Within second day, remove perfect medium, use PBS buffer solution for cleaning, fix 30 minutes by 4% paraformaldehyde room temperature, wash 3 times with PBS damping fluid, add DAPI dyestuff lucifuge dyeing 15 minutes, wash 5 times with PBS damping fluid, in hole, stay 100 μ LPBS damping fluids, with high intension systematic observation fluorescence.
Embodiment 3:
GLP-1R/U2OS cell expressing GLP-1 receptor active is analyzed:
GLP-1R/U2OS cell is incubated at 96 well culture plates by 100 μ L/ holes (1000 cells/well), with the DMEM substratum containing 10% (volume fraction) calf serum, 37 DEG C, 5%CO
2under condition, cultivate 24 hours.Within second day, remove perfect medium, use PBS buffer solution for cleaning, add sample to be tested Exendin-4, room temperature is placed 20 minutes, removes solution in hole, fix 30 minutes by 4% paraformaldehyde room temperature, wash 3 times with PBS damping fluid, add DAPI dyestuff lucifuge dyeing 15 minutes, wash 5 times with PBS damping fluid, in hole, stay 100 μ LPBS damping fluids, by high intension systematic observation change in fluorescence.Result shows generation fluorescence spot in cell, and the U2OS cell of untransfected GLP-1R gene does not have any variation.Above experimental result confirms that the GLP-1R albumen of restructuring GLP-1R/U2OS cell expressing has the physiologically active of natural GLP-1R protein similar, has specificity.
Embodiment 4:
Insulin secretion accelerating peptide Determination of biological activity:
Get the insulin secretion accelerating peptide Exendin-4 of different concns, with the GLP-1R/U2OS cell in embodiment 3 method stimulating growths, each sample is analyzed in high intension systematic observation stimulates the fluorescence spot average intensity after GLP-1R/U2OS cell.Taking concentration of specimens as Transverse coordinate, taking spot average intensity as ordinate zou, make curve.Result represents, after Exendin-4 stimulates, spot average intensity curve is typical anti-S type, and the Exendin-4 of different concns stimulates the average intensity that forms spot after GLP-1R/U2OS cell to have dosage effect dependency.
Claims (7)
1. a strain GLP-1R/U2OS cell strain, it is characterized in that being obtained by following method: build human glucagon-like-peptide-1 acceptor fluorescent protein carrier for expression of eukaryon GLP-1R/pCMV6, and stable transfection human osteosarcoma U2OS cell, obtain a strain GLP-1R/U2OS cell strain.
2. the fluorescin XFP that GLP-1R/U2OS cell strain according to claim 1 contains is GFP, YFP, RFP or any albumen with photoluminescent property.
3. GLP-1R/U2OS cell strain according to claim 1 or other applicable cell are as HEK293 cell, it is characterized in that being obtained by following method: first build human glucagon-like-peptide-1 acceptor GLP-1R green fluorescent protein GFP carrier for expression of eukaryon GLP-1R/pCMV6, carry out transfection U2OS cell by Lipofectamine2000 process specifications again, G418 pressurization screening 2 weeks, acquisition has the positive cell clone of antibiotics resistance, amplification cultivation is the U2OS clone of stably express people GLP-1R, called after GLP-1R/U2OS cell strain.
4. measure the bioactive method of GLP-1 analogue with GLP-1R/U2OS cell strain claimed in claim 1, it is characterized in that: transfection under the GLP-1R gene condition of XFP mark, under receptor stimulant stimulates, induce and in GLP-1R/U2OS cell, produce fluorescence spot, the GLP-1R stably express of transfection.
5. method as claimed in claim 4, it is characterized in that described receptor stimulant is GLP-1 analogue, the activity that produces fluorescence spot intensity and receptor stimulant after GLP-1 binding of receptor and ligand in cell is proportionate, analyze and measure fluorescence spot intensity in GLP-1R/U2OS cell with high intension systematic observation, measure the biological activity of receptor stimulant.
6. method as claimed in claim 4, is characterized in that described GLP-1 analogue is insulin secretion accelerating peptide Exendin-4.
7. method as claimed in claim 4, it is characterized in that the expression result with high intension systematic observation GLP-1R, comprise GLP-1 receptor stimulant, antagonist and conditioning agent etc. with fluorescence spot average intensity change detection GLP-1 analogue in GLP-1R/U2OS cell and GLP-1R bio-active substance.
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