生物工程学报 25 July 2009, 25(7):1088～1094
基于哺乳动物单杂交技术ERα调节剂高通量筛选模型的建立及应用
张倩1, 水小溪1, 范玉玲2, 郝伟丽1, 郑智慧1,2, 路新华2, 赵宝华1, 张华2, 贺建功2
1 河北师范大学生命科学学院, 石家庄050016
2 华北制药集团新药开发有限责任公司, 石家庄050013
摘 要: 雌激素受体α (Estrogen receptor α, ERα)是一种类固醇核受体, 在机体的多种生理功能中起关键作用。为了筛选新的ERα调节剂, 本研究建立一个基于哺乳动物单杂交的报告基因技术的高通量ERα调节剂筛选模型。利用RT-PCR技术从脂肪组织总RNA中扩增ERα配体结合区(Ligand binding domain, ERα LBD)基因序列, 并插入含GAL4 DNA 结合域的pBIND-GAL4表达质粒构建pBIND-GAL4-ERα(LBD)的嵌合表达质粒。该质粒与本室已构建好的含GAL4响应元件和荧光素酶的报告质粒pGL3-GAL4共转染, 通过测定荧光素酶的活性评价ER调节剂的转录调剂的活性。经过多种条件优化, 激动剂阳性药对照雌二醇可以剂量依赖地诱导荧光素酶的表达, 最大上调倍增数可达28.1倍, EC50为0.17 ↘mol/L。拮抗剂阳性对照它莫昔芬可以有效地拮抗雌二醇的活性, 最大下调倍数为6.3倍, EC50为0.1↘ mol/L。该筛选模型可微量化于384孔板, 且Z'因子均大于0.5。利用该模型从2000多个微生物和植物来源的天然产物以及合成化合物中筛选得到4个ERα激动剂。该模型灵敏、稳定, 可以快速进行多种来源的ERα调节剂的筛选和活性评价。
关键词: ERα, 激动剂, 拮抗剂, 共转染, 高通量筛选
Development and application of a mammlian one hybrid-based high-throughput screening model for Erα modulator
Qian Zhang1, Xiaoxi Shui1, Yuling Fan2, Weili Hao1, Zhihui Zheng1,2, Xinhua Lu2, Baohua Zhao1, Hua Zhang2, and Jiangong He2
1 College of Life Sciences, Hebei Normal University, Shijiazhuang 050016, China
2 Drug Research & Development Center of North China Pharmaceutical Group Corporation, National Microbial Medicine Engineering &Research Center, Shijiazhuang 050013, China
Abstract: Estrogen Receptor (ERα) is a member of superfamily of ligand-activated transcription factors which play critical roles in many biological processes. To screen novel modulators of ERα for drug development and biological function research, we developed a mammalian one-hybrid-based high-throughput screening model for ER? modulator. We cloned the ER? LBD gene from the total mRNA of fat tissue by RT-PCR and fused it with the GAL4 DNA binding domain of pBIND-GAL4 plasmid to construct a chimara expression plasmid pBIND-GAL4-Er?(LBD). The L02 cells was cotransfected with pBIND-GAL4-ER?(LBD) and a GAL4-responsive luciferase reporter plasmid pGL3-GAL4, and following treatment with test compounds for 24 h, the activities of luciferase were detected to evaluate the transactivities of ER? modulators. After manner optimizations of transfection conditions, Estradiol, an agonist control, induced the expression of luciferase in a dose-dependent with EC50 of 0.17 ↘mol/L, the maximum folds of induction was about 28.1. Tamoxifen, an antagonist control, efficiently suppressed the estradiol-mediated luciferase induction with EC50 of 0.10 ↘mol/L. Using this screening model, we discovered four ERα agonists from 2000 natural and synthetic compounds.
Keywords: ERα, agonist, antagonist, cotransfection, high-throughput screening
生物谷推荐全文下载：基于哺乳动物单杂交技术ERα调节剂高通量筛选模型的建立及应用
更多全文请查看链接：http://journals.im.ac.cn
声明：本文由《微生物学报》授权生物谷 www.bioon.com 网站发布，如需转载请直接与中国科学院微生物研究所期刊联合编辑部联系并支付相应费用，未经授权不得转载，若转载将付相应的法律责任。
