ERβ重组质粒的构建及其在结肠癌细胞株Caco-2中的表达

Recombination of ERβ eukaryotic expression vector and its expression in Caco-2 cells

目的:基因重组技术构建重组质粒pEGFP-C1-ERβ并检测其在结肠癌细胞株Caco-2中的表达.方法:应用RT-PCR从人结肠癌手术患者正常切缘组织中分离、扩增目的基因片段,将所得cDNA定向克隆到真核表达载体pEGFP-C1中,采用双酶切和测序分析鉴定插入基因的序列;脂质体介导重组质粒pEGFP-C1-ERβ瞬时转染Caco-2并上流式细胞仪分选,获得比较单一的转染细胞;分别采用RT-PCR、Western blot检测转染前后ERβ基因不同分子水平表达.结果:酶切鉴定和测序分析表明重组表达质粒pEGFP-C1-ERβ构建无误;RT-PCR和Western blot分析均表明,与转染空质粒pEGFP-C1组和空白对照组细胞相比,转染重组表达质粒pEGFP-C1-ERβ组细胞ERβ基因表达水平明显提高.结论:成功构建重组质粒pEGFP-C1-ERβ并在Caco-2细胞株中表达,为进一步研究ERβ如何通过雌激素受体通路调控下游靶基因表达和参与结肠癌表遗传学发生机制奠定了基础。

AIM： To construct the recombinant plasmid pEGFP-C1-ERβ with gene recombinant technique and detect its expression in Caco-2 cells. METHODS： ERβ gene in total RNA was isolated from human normal colon tissue of colorectal cancer patients and its segments were amplified by RT-PCR. The obtained cDNA was cloned into eukaryotic expression vector pEGFP-C1 to generate recombinant pEGFP-C1-ERβ. Sequence of the inserted gene was identified and analyzed after restriction enzyme digestion. Liposomemediated recombinant plasmid pEGFP-C1-ERβ was transfected into Caco-2 cells and identified by flow cytometry （FCM）. RT-PCR and Western blot were used to detect expression of the ERβ gene at molecular level before and after transfection. RESULTS： Recombinant pEGFP-C1-ERβ was confirmed by restriction enzyme digestion and sequence analysis. The expression of Caco-2 cells transfected with pEGFP-C1-ERβ was higher than that of other controls. CONCLUSION： Recombinant pEGFP-C1-ERβ can be successfully constructed and expressed in Caco-2 cells, which lays a foundation for further study on ERβ gene in carcinogenesis by regulating the expression of down stream target gene through estrogenic hormone receptor.