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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

A technology of GLP-1 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 is expensive and other issues to achieve the effect of reducing false positives, reducing detection costs, and low cost

Inactive Publication Date: 2014-08-06
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 screening peptide and non-peptide GLP-1 (Glucagon-Like Peptide 1) analogs as well as preparation method and application of cell line
  • 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
  • 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

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Construction of GFP fluorescently labeled human GLP-1R eukaryotic expression vector GLP-1R / pCMV6, establishment of GLP-1R / U2OS cell line, and determination of the biological activity of GLP-1 analogues by the change in the average intensity of fluorescent spots in GLP-1R / U2OS cells .

[0025] 1. Construction of GFP fluorescently labeled human GLP-1R eukaryotic expression vector GLP-1R / pCMV6:

[0026] 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 full length of 1392bp.

[0027] Its upstream primer: 5'-gaggcgatcgccATGGCCGGCGCCCCCGGC-3';

[0028] Downstream primer: 5'-gcgacgcgtGCTGCAGGAGGCCTGG CAAG-3'.

[0029] 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.

[0030] 3. Digest plasmid G-D (pCMV6-AC-GFP-DR5) with restriction enzymes Sg...

Embodiment 2

[0043] Expression of GLP-1R on GLP-1R / U2OS cell membrane:

[0044] 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

[0046] GLP-1R / U2OS cell expression GLP-1 receptor activity analysis:

[0047] 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 / U2OS cells h...

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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: [0001] 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: [0002] Incretins are hormones released from the gastrointestinal tract during nutrient absorption to increase insulin secretion. The two intestinal peptides that account for most of the effects of incretin are: 1) GLP-1 (Glucagon-like peptide, glucagon-like peptide 1): encoded by the proglucagon gene, stimulated by nutrients A hormone conta...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/85C12Q1/02G01N21/64C12R1/91
CPCG01N33/5044C12N5/0693C12N15/85C12N2510/00C12N2503/00
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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