ERβ表达载体的构建及其在肿瘤细胞中的功能研究

Construction of ERβ expression vector and its function in different cancer cells

目的 构建ERβ表达载体 ,探讨其在肿瘤细胞株中的表达及功能。 方法 利用常规PCR技术扩增人全长ERβ编码序列 ,并将其克隆到真核表达载体pCDNA3中 ,得到重组质粒pCDNA3 ERβ。用Westernblot和体外翻译方法检测ERβ的表达情况。将pCDNA3 ERβ分别转染SV4 0病毒转化的胚胎肾细胞 2 93T、乳腺癌细胞MDA MB 4 35、MDA MB 4 36、SKBR3和前列腺癌细胞PC 3,用含雌激素应答元件的报告基因系统检测ERβ在这些不同细胞中的功能。 结果 经酶切鉴定 ,证实重组质粒pCDNA3 ERβ含有目的基因ERβ。Westernblot检测表明 ,转染pCDNA3 ERβ的细胞表达了相对分子量为 6 30 0 0的ERβ蛋白。体外翻译进一步证实该表达载体翻译了ERβ蛋白。报告基因系统检测表明 ,ERβ在不同肿瘤细胞中的表达均激活了雌激素应答的ERE和C3报告基因的表达。 结论 pCDNA3 ERβ在肿瘤细胞中既可表达 ,又具有功能活性 ,为进一步研究ERβ在肿瘤细胞中的作用奠定了基础。

Objective To construct an ERβ expression vector and study its expression and function in different cancer cells. Methods Standard PCR was used to amplify the full length coding sequence of ERβ. The amplified ERβ gene was cloned into the eukaryotic expression vector pCDNA3, generating pCDNA3 ERβ. The ERβ expression was detected by Western blot and in vitro translation. The biological activity of ERβ was detected by transfecting the pCDNA3 ERβ into SV40 transformed embryonic kidney cell line 293T,breast cancer cell lines MDA MB 435?MDA MB 436? SKBR3, and prostate cancer cell line PC 3, with reporters containing estrogen response elements. Results The recombinant plasmid pCDNA3 ERβ was confirmed by restriction analysis to contain the ERβ gene. The 63 000 ERβ expression was shown by Western blot and further confirmed by in vitro translation. The ERβ expression in different cancer cells was demonstrated to stimulate the expression of the reporters containing estrogen response elements, ERE and C3. Conclusion ERβ protein is successfully expressed and has biological activity ,laying solid foundation for further study on its role in cancer cells. [