生物工程學(xué)報(bào) 25 July 2009, 25(7):1088～1094

基于哺乳動(dòng)物單雜交技術(shù)ERα調(diào)節(jié)劑高通量篩選模型的建立及應(yīng)用

張倩1, 水小溪1, 范玉玲2, 郝偉麗1, 鄭智慧1,2, 路新華2, 趙寶華1, 張華2, 賀建功2

1 河北師范大學(xué)生命科學(xué)學(xué)院, 石家莊050016

2 華北制藥集團(tuán)新藥開發(fā)有限責(zé)任公司, 石家莊050013

摘 要: 雌激素受體α (Estrogen receptor α, ERα)是一種類固醇核受體, 在機(jī)體的多種生理功能中起關(guān)鍵作用,。為了篩選新的ERα調(diào)節(jié)劑, 本研究建立一個(gè)基于哺乳動(dòng)物單雜交的報(bào)告基因技術(shù)的高通量ERα調(diào)節(jié)劑篩選模型,。利用RT-PCR技術(shù)從脂肪組織總RNA中擴(kuò)增ERα配體結(jié)合區(qū)(Ligand binding domain, ERα LBD)基因序列, 并插入含GAL4 DNA 結(jié)合域的pBIND-GAL4表達(dá)質(zhì)粒構(gòu)建pBIND-GAL4-ERα(LBD)的嵌合表達(dá)質(zhì)粒,。該質(zhì)粒與本室已構(gòu)建好的含GAL4響應(yīng)元件和熒光素酶的報(bào)告質(zhì)粒pGL3-GAL4共轉(zhuǎn)染, 通過測(cè)定熒光素酶的活性評(píng)價(jià)ER調(diào)節(jié)劑的轉(zhuǎn)錄調(diào)劑的活性,。經(jīng)過多種條件優(yōu)化, 激動(dòng)劑陽性藥對(duì)照雌二醇可以劑量依賴地誘導(dǎo)熒光素酶的表達(dá), 最大上調(diào)倍增數(shù)可達(dá)28.1倍, EC50為0.17 ↘mol/L。拮抗劑陽性對(duì)照它莫昔芬可以有效地拮抗雌二醇的活性, 最大下調(diào)倍數(shù)為6.3倍, EC50為0.1↘ mol/L,。該篩選模型可微量化于384孔板, 且Z'因子均大于0.5,。利用該模型從2000多個(gè)微生物和植物來源的天然產(chǎn)物以及合成化合物中篩選得到4個(gè)ERα激動(dòng)劑。該模型靈敏,、穩(wěn)定, 可以快速進(jìn)行多種來源的ERα調(diào)節(jié)劑的篩選和活性評(píng)價(jià),。

關(guān)鍵詞: ERα, 激動(dòng)劑, 拮抗劑, 共轉(zhuǎn)染, 高通量篩選

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

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