Cell strain for measuring bioactivity of GLP-1 and functional analogue thereof and application of cell strain

A GLP-1 and GLP-1R technology, applied in the field of drug biological activity detection, can solve the problems of difficult standardization of detection methods, poor data repeatability, and difficult to obtain, and achieves good application prospects, low cost, and easy standardization.

Inactive Publication Date: 2012-08-29
无锡和邦生物科技有限公司 +1
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The detection method has the disadvantages that experimental animals are not easy to obtain, the operati

Method used

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  • Cell strain for measuring bioactivity of GLP-1 and functional analogue thereof and application of cell strain
  • Cell strain for measuring bioactivity of GLP-1 and functional analogue thereof and application of cell strain
  • Cell strain for measuring bioactivity of GLP-1 and functional analogue thereof and application of cell strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Embodiment 1: Construction of recombinant GLP-1R / pcDNA3.1 (+) eukaryotic expression plasmid

[0029] 1.1 Amplification and identification of GLP-1R gene

[0030] A pair of gene-specific primers were designed according to the cDNA sequence (SEQ ID NO: 1) of the open reading frame encoding human GLP-1R protein:

[0031] GLP-1R1: 5'-AAAAGCTTATGGCCGGCGCCCCCGG-3' (SEQ ID NO: 3);

[0032] Hind III

[0033] GLP-1R2: 5'-TTCTCGAGTCAGCTGCAGGAGGCCTG-3' (SEQ ID NO:4)

[0034] Xho I

[0035] The 5' end of primer GLP-1R1 has Hind III restriction endonuclease site, the 5' end of GLP-1R2 has Xho I restriction endonuclease sites. Using the GLP-1R coding gene (pReceiver-M02 plasmid DNA, provided by the Scientific Research Department of the Jiangsu Schistosomiasis Control Institute) as a template, PCR amplification was performed. The specific conditions are as follows: In a 0.2 mL PCR tube, add 1 μL of pReceiver-M02 plasmid DNA; 10×Taq Buffer, 10 μL; ...

Embodiment 2

[0040] Example 2: Construction and identification of genetically engineered cell lines expressing GLP-1R protein

[0041] 2.1 HEK293 cells transfected with recombinant GLP-1R / pcDNA3.1(+) plasmid

[0042] HEK293 cells were seeded in six-well plates (4 × 10 5 cells / well), each well contains 100 μL DMEM medium (without antibiotics), cultured for 18~24h to 80% cell density. Mix 2 μg of the linearized recombinant plasmid with 3 μL of FuGene 6 for cell transfection.

[0043] After 24 hours of transfection, the transfected cells were subcultured into fresh culture medium containing 300 μg / mL G418 (the initial concentration of G418 was determined by the preliminary experiment) at a ratio of 1:10, and placed at 37°C, 5% CO 2 cultured in an incubator. The medium was changed every two days, and most of the cells were observed to die after 48-72 hours of culture. The surviving cells were transferred to another six-well plate, and the concentration of G418 was gradually increased (up t...

Embodiment 3

[0049] Example 3: GLP-1R / HEK293 cell expression GLP-1 receptor activity analysis

[0050] When the GLP-1 receptor protein expressed by recombinant genetically engineered GLP-1R / HEK293 cells has physiological activity, when it is stimulated by the agonist GLP-1 or its functional analogues, the physiological metabolic activities of GLP-1R / HEK293 cells Enhanced, manifested as an increase in intracellular cAMP content. Therefore, whether the GLP-1R protein expressed by GLP-1R / HEK293 cells has physiological activity can be determined by measuring the change of cAMP content in GLP-1R / HEK293 cells after being stimulated by agonists such as GLP-1. The specific method is as follows:

[0051] GLP-1R / HEK293 cells were cultured in a 96-well culture plate at 100 μL / well (30,000 cells / well) in DMEM medium at 37°C, 5% CO 2 Conditioned for 24 hours. The complete medium was removed the next day, and the basal medium was added to culture overnight (about 15 hours). Discard the basal medium,...

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Abstract

The invention relates to a cell strain for measuring the bioactivity of GLP-1 and functional analogue thereof and an application of the cell strain and belongs to the technical field of measurement of drug bioactivity. The GLP-1R/HEK293 cell strain is preserved in the common microorganism center of China microorganism culture preservation management committee and the preservation number is CGMCCNo.5909. The invention relates to a measuring method for establishing express glucagon peptide-1 receptor (GLP-1R) genetic engineering cell strain GLP-1R/HEK293 and measuring the bioactivity of GLP-1 and analogue thereof in external stimulation on the basis of the change in the cAMP level in the cell after the GLP-1R/HEK293 cell strain is stimulated by the glucagon peptide-1 receptor (GLP-1R) and the functional analogue thereof. The measuring method is easily standardized, is excellent in repeatability and has the characteristics of low cost, convenience and accuracy, thereby being excellent in application prospect.

Description

technical field [0001] The present invention relates to the establishment of a genetically engineered cell strain expressing glucagon-like peptide-1 receptor protein, and the change of cAMP level after the cell strain is stimulated by glucagon-like peptide-1 and its functional analogs The invention discloses a detection method for determining the biological activity of glucagon-like peptide-1 and its functional analogues, and belongs to the technical field of drug biological activity detection. Background technique [0002] Glucagon-like peptide-1 (Glucogan like Peptide-1, GLP-1) acts on the glucagon-like peptide-1 receptor (Glucogan like Peptide-1 Receptor, GLP-1R) of islet β cells to promote insulin gene The transcription, synthesis and secretion of insulin, and can stimulate the proliferation and differentiation of islet β cells, inhibit the apoptosis of islet β cells, and increase the number of islet β cells. Studies have proved that GLP-1 can significantly improve the ...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12Q1/02C12R1/91
Inventor 杨建良王军志饶春明陆晶王兰
Owner 无锡和邦生物科技有限公司
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