Livin shRNA稳定转染Hep-2细胞系的建立

Construction of eukaryotic expression plasmid of Livin shRNA and establishment of their stably transfected Hep-2 cell lines

目的构建靶向Livin基因干扰真核表达质粒,建立干扰质粒稳定转染的Hep-2细胞系,下调Livin基因在Hep-2细胞系中的表达。方法针对Livin基因的mRNA序列设计干扰序列,分别构建1个shRNA质粒表达载体和1个阴性对照质粒,大肠杆菌扩增、酶切及测序鉴定,用脂质体介导转染Hep-2细胞,经G418筛选出稳定转染的细胞系,用Real-time PCR和Western blot检测Livin在mRNA和蛋白水平的抑制效果。结果经测序证实成功构建pGenesil-Livin shRNA真核表达质粒。干扰质粒稳定转染的Hep-2细胞在荧光显微镜下观察,发出绿色荧光。重组质粒转染Hep-2细胞后,Livin基因在mRNA及蛋白水平明显下降,mRNA水平下调47.17%,蛋白水平下调34.25%。结论成功构建以Livin为靶向的Livin shRNA真核表达质粒。对喉癌Hep-2细胞中Livin的表达具有显著抑制效应,为研究Livin在Hep-2中的功能和喉癌的基因治疗奠定了基础。

OBJECTIVE To construct interference eukaryotic expression plasmid of Livin gene, to establish Hep-2 cell line with stable transfected recombinant plasmids, and to reduce the expression of Livin in Hep-2 cell line. METHODS Targeting Livin gene sequence, a plasmid expression vectors coding for shRNA and a control vector containing random DNA fragment were constructed. The recombinant plasmids were amplified in E.coli, then identified by restriction digestion, and sequencing. The vectors were transfected into Hep-2 cells by Lipofectamine. The cells containing stable transformants were selected by the ability of resistance to G418. Livin expression was assayed by real-time quantitative PCR and Western blot. RESULTS The successful construction of pGenesil-1-Livin was confirmed by DNA sequencing. Green fluorescence of the stable transfected Hep-2 cell line could be observed under fluorescence micorscope. Transfection of Livin-shRNA plasmids significantly down-regulated Livin expression in Hep-2 cells at both mRNA level and protein level, with an inhibition ratio of 47.17% at the mRNA level and 34.25% at the protein level respectively. CONCLUSION pGenesil-l-Livin has been successfully constructed and it can down-regulate Livin expression after transfected into Hep-2 cells, which could facilitate further studies of Livin functions in Hep-2 cell line and its application in laryngeal carcinoma gene therapy.