表达Cys548雌激素受体突变型cDNA的真核载体构建及其促转录活性分析

目的 考察一个548位点C→T单核苷酸突变的雌激素受体(ER)在体外条件下,对乳腺癌细胞株(MCF-7)信号通路的影响,以探讨ER单核苷酸突变引起非雌激素依赖性性早熟的可能机制.方法 用重叠延伸PCR定点诱变技术对548位点的碱基进行定点突变.构建定点突变表达载体pSG5-MuER.构建含雌激素受体反应元件(ERE)的萤光素酶报告基因载体pGL3-ERE-Luc.将野生和突变ER质粒分别和pGL3-ERE-Luc共转染MCF-7细胞,观察萤光素酶的变化,以检测突变ER反应活性的变化.结果 成功构建ER突变质粒psG5-MuER和ER报告质粒pGL3-ERE-Luc,psG5-MuER较pSG5-ER能够增加萤光素酶的产生.结论 该突变雌激素受体在体外具有高促转录活性特征,构建的载体可用于进行进一步的相关研究.

Construction of mutant of ER,ER response vector and effects of its transcriptional activity

Objective To observe whether a 548 point C→T single nucleotide mutation of estrogen receptor (ER) affects breast cancer cells (MCF-7) signaling pathway in vitro, and to explore possible mechanism of this ER single nucleotide mutation inducing non-estrogen-dependent precocious puberty.Methods ER gene was inserted into wild-ER pSG5 plasmid as a template using site-directed mutagenesis overlap extension PCR technology to the base 548 points for site-directed mutagenesis. Expression vector of site-directed mutagenesis pSGS-MuER and the ER response element reports luciferase gene vector pGL3-ERE-Luc were constructed. Wild and mutant plasmids were cotransfected with pGL3-ERE-Luc into MCF-7 cells.Luciferase change was observed in order to detect mutation ER reactivity. Results The ER mutants and the ER response element containing the luciferase report gene vector were successfully constructed, pSG5-MuER increased luciferase production than pSG5-ER. Conclusions The mutations of estrogen receptor in vitro lead to high reactivity characteristics. The vector can be used for further reseach.