A Cell-based High-throughput Screening Assay for Farnesoid X Receptor Agonists

作     者：ZHI-HUI ZHENG Guo-PING LV SHU-YI SI YUE-SHENG DONG BAO-HUA ZHAO HUA ZHANG JIAN-GONG HE 

作者机构：Insitute of Medicinal Biotechnology Peking Union Medical College / Chinese Academy of Medical Sciences Beijing 100050 China College of Life Science Hebei Normal University Shijiazhuang 050016 Hebei China New Drug Research ＆ Development Center of North China Pharmaceutical Group Corporation National Microbial Medicine Engineering ＆ Research Center Shijiazhuang 050015 Hebei China 

出 版 物：《Biomedical and Environmental Sciences》 (生物医学与环境科学（英文版）)

年 卷 期：2007年第20卷第6期

页      面：465-469页

核心收录：

学科分类：0710[理学-生物学] 07[理学] 071009[理学-细胞生物学] 09[农学] 0901[农学-作物学] 090102[农学-作物遗传育种] 

基　　金：supported by the Ministry of Science and Technology, PRC in Mega-projects of Science Research During the 10th Five-Year Plan Period (No. 2004AA2Z38784) National Natural Science Foundation of China (No. 30472026) 

主　　题：Farnesoid X receptor Agonist High-throughput screening Chimera 

摘      要：Objective To develop a high-throughput screening assay for Farnesoid X receptor （FXR） agonists based on mammalian one-hybrid system （a chimera receptor gene system） for the purpose of identifying new lead compounds for dyslipidaemia drug from the chemical library. Methods cDNA encoding the human FXR ligand binding domain （LBD） was amplified by RT-PCR from a human liver total mRNA and fused to the DNA binding domain （DBD） of yeast GAL4 of pBIND to construct a GAL4-FXR （LBD） chimera expression plasmid. Five copies of the GAL4 DNA binding site were synthesized and inserted into upstream of the SV40 promoter of pGL3-promoter vector to construct a reporter plasmid pG5-SV40 Luc. The assay was developed by transient co-transfection with pG5-SV40 Luc reporter plasmid and pBIND-FXR-LBD （189-472） chimera expression plasmid. Results After optimization, CDCA, a FXR natural agonist, could induce expression of the luciferase gene in a dose-dependent manner, and had a signal/noise ratio of 10 and Z＇ factor value of 0.65, Conclusion A stable and sensitive cell-based high-throughput screening model can be used in high-throughput screening for FXR agonists from the synthetic and natural compound library.