STAT3-shRNA-pGenesil稳定表达载体的构建与应用

The Construction and Application of STAT3-shRNA-pGenesil Expression Vector

目的:构建siRNA的DNA稳定表达载体,为研究RNAi在哺乳动物内持续、稳定抑制靶基因表达奠定基础。方法:合成含靶向STAT3基因的siRNA转录模板的发夹结构,将载体质粒pGenesil-1用BamHⅠ与HindⅢ进行双酶切后,T4DNA连接酶连接成重组质粒,转染到肝癌SMMC-7721细胞中进行稳定筛选、表达,检测稳定筛选前后STAT3基因的表达变化。结果:重组质粒在大肠杆菌菌株JM109内扩增。提纯、纯化后经PCR及测序鉴定证明STAT3-siRNA转录模板完整、正确地插入到pGenesil-1质粒中,建立了稳定抑制STAT3基因的SMMC-7721细胞株,并在mRNA水平抑制了肝癌SMMC-7721细胞STAT3基因表达。结论:成功构建了STAT3-shRNA-pGenesil稳定表达载体,能在哺乳动物细胞中表达,并初步应用于靶基因的抑制。

Objective：To construct DNA stable expressed vector of siRNA for establishing material foundation of gene expression of long inhibition effectively by RNAi in mammalian cells. Methods ：The hairpin structure of siRNA transcript template targeting STAT3 gene was synthesized. Then, both pGenesil - 1 empty vector and siRNA transcript template were digested by BamH I and HindIII. Two digested fragments were ligated with T4 DNA ligase to recombinant vector. The recombinant vector was then transfected into hepatocarcinoma SMMC- 7721 cells to selected stably and detected the expression alteration of STAT3. Results：The recombination plasmid was amplified in the E. coll. JM 109. After the identification of PCR and sequencing, the reconstructive plasmid was confirmed that contained the correct and full nucleotide sequence of STAT3 - siRNA transcript template in pGenesil - 1 vector. The mRNA of STAT3 gene were inhibited by RNAi in hepatocarcinoma SMMC - 7721 cells. Conclusion：The STAT3 - shRNA - pGenesil expression vector was constructed successfully, could express in mammalian cells, and initiate employment of suppressing target gene.