华东师范大学学报(自然科学版) ›› 2007, Vol. 2007 ›› Issue (4): 112-118.

• 生命科学 • 上一篇    下一篇

中草药中胰高血糖素受体拮抗剂的高通量细胞筛选

万 娟， 许治良   

  1. 华东师范大学 生命科学学院,上海市脑功能基因组学重点实验室,上海 200062
  • 收稿日期:2006-03-01 修回日期:2006-04-05 出版日期:2007-07-25 发布日期:2007-07-25
  • 通讯作者: 许治良

High Throughput Screening Method of Identifying Antagonist for Glucagaon Receptor in Chinese Herbs(Chinese)

WAN Juan, XU Zhi-liang
   

  1. Key Laboratory of Brain Functional Genomics of Shanghai, School of Life Science, East China NormalUniversity, Shanghai 200062, China
  • Received:2006-03-01 Revised:2006-04-05 Online:2007-07-25 Published:2007-07-25
  • Contact: XU Zhi-liang

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.

关键词: 胰高血糖素受体, 报告基因, 拮抗剂, 高通量药物筛选, 胰高血糖素受体, 报告基因, 拮抗剂, 高通量药物筛选

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.

Key words: reporter gene, antagonist, high throughput screening, glucagon receptor, reporter gene, antagonist, high throughput screening

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