雌激素受体α真核表达载体的构建及其转录活性

Construction of Estrogen Receptor α Eukaryotic Expression Vector and Detection of its Transcription Activity

目的:构建雌激素受体α(ERα)高效真核表达载体,并检测其转录活性。方法:用常规PCR方法特异扩增带有Flag标签的人ERα全长编码序列,经双酶切后将其克隆到真核表达载体pIRESpuro2中,构建重组质粒pIRESpuro2-Flag-ERα;用Westernblot鉴定该重组质粒介导的Flag-ERα在人胚肾细胞株293T中的表达情况;用含雌激素应答元件的报告基因系统检测ERα的转录活性。结果:构建了pIRESpuro2-Flag-ERα重组质粒,该质粒介导的融合蛋白在293T细胞中得到表达,并可以激活含雌激素应答元件的报告基因的活性。结论:ERα高效真核表达载体的构建为进一步研究ERα在乳腺癌中的作用奠定了基础。

Objective：To construct estrogen receptor α（ERα） eukaryotic expression vector,and detect its transcriptional activity.Methods：The coding sequence of ERα with Flag tag were amplified by standard PCR,digested with two enzymes,and cloned into pIRESpuro2 vector,to generate the corresponding recombinant plasmid pIRESpuro2-Flag-ERα.Western blot was performed to determine the expression of the plasmid-directed Flag-ERα in 293T cells.A reporter containing estrogen response element was used to detect the transcriptional activity of Flag-ERα.Results：The recombinant plasmid was successfully constructed.Expression of Flag-ERα in 293T cells stimulated the reporter gene expression.Conclusion：The construction of the ERα eukaryotic expression vector lays foundation for further functional study of ERα.