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.
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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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