RNAi-DNA表达载体的构建与应用

The construction and application of siRNA-DNA expression vector

目的构建siRNA的DNA表达载体,为研究RNAi在哺乳动物内抑制靶基因表达奠定基础。方法合成含靶向EGFP基因的siRNA转录模板的发夹结构,将载体质粒pTZU6+1用Sal I+Xba I进行双酶切后,T4DNA连接酶连接成重组质粒,转染到肝癌SMMC-7721细胞中进行表达,检测转染前后EGFP表达的变化。结果重组质粒在大肠杆菌菌株JM109内扩增。提纯、纯化后用HindⅢ、EcoRI酶切鉴定及测序鉴定证明EGFP-siRNA转录模板完整、正确的插入到pTZU6+1质粒中,并在mRNA和蛋白水平抑制了肝癌SMMC-7721细胞中的EGFP表达。结论成功构建了siRNA的DNA表达载体,并初步应用于靶基因的抑制。

Objeetive To construct DNA expression vector of siRNA,and to study the target gene expression inhibition by RNAi in mammalian cells. Methods The hairpin structure of siRNA transcript template targeting EGFP gene was first synthesized. Then, both PTZU6＋1 empty vector and siRNA transcript template were digested with SalⅠ/XbaⅠ, Two digested fragments were ligated with T4 DNA ligase. The recombinant vector was then transfected into hapartocarcinoma SMMC-7721 cells and the expression alteration of EGFP was detected. Results The recombination plasmid was amplified in the E. coli. JM109. The identification of redigesting with HindⅢ /EcoRⅠ and sequencing showed that the reconstructive plasmid contained the correct and full nucleotide sequence of EGFP-siRNA transcript template. The mRNA and protein of EGFP gene were inhibited by RNAi in hapartocarcinoma SMMC-7721 cells. Conclusion The DNA expression siRNA vector was constructed successfully and could be used to suppress target gene.