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

Development and application of a mammlian one hybrid-based high-throughput screening model for Erα modulator

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 mmol/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 mmol/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。

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 ERα modulator. We cloned the ERα 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-Erα(LBD). The L02 cells was cotransfected with pBIND-GAL4-ERα(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 ERα 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 mmol/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 mmol/L. Using this screening model, we discovered four ERα agonists from 2000 natural and synthetic compounds.