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Cell line for scanning peptide and non-peptide GLP-1 (Glucagon-likepeptide-1) analogues as well as preparation method and application thereof

A technology of GLP-1R and GLP-1R-, which is used in the field of screening peptide and non-peptide GLP-1 analogs of cell lines and their preparation and application, which can solve the problem of immune response, increase the economic burden of patients, and reduce the price Expensive and other issues, to achieve the effect of reducing false positives, reducing detection costs, and low cost

Pending Publication Date: 2017-09-26
KUNMING BEIERJI SCI & TECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, since these agonists introduce foreign proteins into the human body, they may trigger the body's immune response
Currently marketed GLP-1 analogues are expensive and will increase the financial burden on patients

Method used

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  • Cell line for scanning peptide and non-peptide GLP-1 (Glucagon-likepeptide-1) analogues as well as preparation method and application thereof
  • Cell line for scanning peptide and non-peptide GLP-1 (Glucagon-likepeptide-1) analogues as well as preparation method and application thereof
  • Cell line for scanning peptide and non-peptide GLP-1 (Glucagon-likepeptide-1) analogues as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1 Construction of GFP fluorescence-labeled human GLP-1R eukaryotic expression vector GLP-1R / pCMV6, establishment of GLP-1R / U2OS cell line, and determination of the average intensity change of fluorescent spots in GLP-1R / U2OS cells GLP-1 is similar biological activity.

[0033] 1. Construction of GFP fluorescently labeled human GLP-1R eukaryotic expression vector GLP-1R / pCMV6:

[0034] Human GLP-1R cDNA amplification: using human GLP-1R cDNA as a template, the full-length human GLP-1R cDNA coding region was amplified by PCR, with a total length of 1392bp.

[0035] Its upstream primer: 5'-gaggcgatcgccATGGCCGGCGCCCCCGGC-3';

[0036] Downstream primer: 5'-gcgacgcgtGCTGCAGGAGGCCTGG CAAG-3'.

[0037] 2. Recover and purify the PCR product, connect it to pMD18-T Vector to obtain the recombinant plasmid GLP-1R / pMD18-T, and use restriction enzymes Sgf I and Mlu I to digest the plasmid.

[0038] 3. Digest plasmid G-D (pCMV6-AC-GFP-DR5) with restriction enzymes Sgf I a...

Embodiment 2

[0050] Example 2 Expression of GLP-1R on GLP-1R / U2OS cell membrane

[0051] GLP-1R / U2OS cells were cultured in 96-well culture plates at 100 μL / well (1000 cells / well) in DMEM medium containing 10% (volume fraction) calf serum at 37°C and 5% CO 2 Conditioned for 24 hours. The next day, the complete medium was removed, washed with PBS buffer, fixed with 4% paraformaldehyde at room temperature for 30 minutes, washed 3 times with PBS buffer, stained with DAPI dye in the dark for 15 minutes, washed 5 times with PBS buffer, Leave 100 μL PBS buffer in the well, and observe the fluorescence with the high content system.

Embodiment 3

[0052] Example 3 GLP-1R / U2OS cell expression GLP-1 receptor activity analysis

[0053] GLP-1R / U2OS cells were cultured in 96-well culture plates at 100 μL / well (1000 cells / well) in DMEM medium containing 10% (volume fraction) calf serum at 37°C and 5% CO 2 Conditioned for 24 hours. The next day, remove the complete medium, wash with PBS buffer, add Exendin-4 to the sample to be tested, let it stand at room temperature for 20 minutes, remove the solution in the well, fix with 4% paraformaldehyde at room temperature for 30 minutes, wash with PBS buffer for 3 Add DAPI dye for 15 minutes in the dark, wash 5 times with PBS buffer, leave 100 μL PBS buffer in the well, and observe the fluorescence change with the high-content system. The results showed that fluorescent spots were produced in the cells, while the U2OS cells not transfected with GLP-1R gene did not have any changes. The above experimental results confirm that the GLP-1R protein expressed by the recombinant GLP-1R / U2O...

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Abstract

The invention provides a cell line for scanning peptide and non-peptide GLP-1 (Glucagon-like Peptide-1) analogues as well as a preparation method and application thereof and belongs to the technical field of high-flux screening and detection of medicines. The invention relates to the detection device for stably expressing the establishment of a fluorescence labeling human glucagon-like peptide-1 receptor (GLP-1R) protein cell line GLP-1R / U2OS, forming fluorescence spots in cells after the GLP-1R / U2OS cell line is stimulated by the GLP-1 analogue Exendin-4 and determining the bioactivity of the GLP-1 analogues by observing form changes through utilizing a high-content system. The detection method provided by the invention is easily standardized, has good repeatability, has the characteristics of GLP-1 receptor specificity, low cost, accuracy and convenience and has a good application prospect.

Description

[0001] This application is a divisional application of Chinese application 201410234121.2 filed on May 29, 2014, entitled "A cell line for screening peptide and non-peptide GLP-1 analogues and its preparation method and application". technical field [0002] The invention belongs to the technical field of high-throughput screening and detection of drugs, and specifically relates to a cell line expressing fluorescently labeled human glucagon-like peptide-1 receptor (GLP-1R) protein cell line GLP-1R / U2OS cell line and its establishment method, and based on the morphological changes of fluorescent spots in the cells after the cell line is stimulated by glucagon-like peptide-1 analogs to determine the biological activity of glucagon-like peptide-1 analogs and its receptor agonists and antagonists Methods for the detection of agents and modulators. Background technique [0003] Incretins are hormones released from the gastrointestinal tract during nutrient absorption to increase ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/85C12Q1/02
CPCC12N5/0693C12N15/85C12N2503/00C12N2510/00G01N33/5044
Inventor 姚洪玉魏云林张敏张宽仁刘东波季秀玲
Owner KUNMING BEIERJI SCI & TECH
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