High Throughput Screening Method of Identifying Antagonist for Glucagaon Receptor in Chinese Herbs(Chinese)
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摘要: 为筛选胰高血糖素受体拮抗剂，建立了一种高通量筛选胰高血糖素受体拮抗剂的细胞模型.利用PCR方法扩增得到人的胰高血糖素受体 cDNA，并连接到哺乳动物细胞表达载体pCDNA3.1上，构成质粒h glucagons-R/pCDNA3.1，将这种质粒与报告基因质粒(3×CRE/3×MRE/SRE-LUC)共转染到HEK293细胞，通过G418筛选，建立稳定的人胰高血糖素受体拮抗剂筛选细胞株.利用胰高血糖素受体内源激动剂探索和优化了筛选条件，筛选方法合理， Z′因子大于0.6，系统稳定，符合胰高血糖素受体拮抗剂的高通量筛选的要求.Abstract: This paper generated a cell-based functional assay for human glucagon receptor antagonist screening. The human glucagon receptor cDNA was cloned into a mammalian expression vector pCDNA3.1 (h Glucagon receptor /pCDNA3.1). This plasmid was co-transfected with a reporter gene construct (3×CRE/3×MRE/SRE-LUC) in HEK293 cells. Stable cell line expressing both human glucagon receptor and the reporter gene was selected for compounds screening. The assay condition was optimized. Our results demonstrated that this cell-based assay can be optimized for high throughput screening and applied to identify antagonist for glucagon receptor in Chinese Herbs.
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