RNAi真核表达载体上报告基因的构建及其在人结肠癌HCT-8细胞中的表达

Construction of RNAi eukaryotic expression vector pSilencer31-H1/GFP a nd its expression in HCT-8 cells

目的:构建携带绿色荧光蛋白报告基因的pSilencer3.1-H1真核表达载体,并转染真核细胞稳定表达,为进一步应用于RNAi相关研究奠定了基础。方法:应用PCR方法从pEGFP-C1标准质粒中获得GFP基因片段,在克隆载体中鉴定、测序;用DNA重组技术将片段定向插入RNAi常用载体pSilencer3.1-H1质粒中,构建pSilencer3.1-H1/GFP重组表达载体,经PCR及酶切鉴定后,通过脂质体介导转染HCT-8真核细胞;荧光显微镜观察GFP在细胞内的表达。结果:真核表达载体pSilenc-er3.1-H1中正确插入了GFP基因片段,并在人结肠癌HCT-8真核细胞中稳定的表达。结论:成功构建了pSilencer3.1-H1/GFP真核表达载体。

Objective To construct the eukaryotic expression plasmid containing green fluorescent prot ein gene and observe its expression in HCT-8 cells,which lays the foundation fo r further development of RNAi related research.Method GFP was obtained from pEGFP-C1 by PCR technology and was inserted into pSilencer3.1-H1.After identified by restriction endonucleas e and sequence analysis,the recombinant expression plasmid was transfected with l ipofectamine 2000 to HCT-8 cells.The expression was observed under a fluorescen t microscope.Results Restr i ctive endonuclease assay and sequence analysis verified the successful construct ion of the recombinant vector pSilencer3.1-H1/GFP,and GFP protein was stable e x pressed in HCT-8 cells.Conclusion The GFP eukaryotic expression plasmid is successfully constructed and ex pressed in HCT-8 cells.