抑制14-3-3ζ表达的siRNA及其重组腺相关病毒载体的构建

Construction of Recombinant Adeno-associated Virus Vector Containing a siRNA Targeting 14-3-3ζ

[目的]通过构建靶向14-3-3ζ的siRNA及其报告基因EGFP的重组腺相关病毒载体,包装出含有目标基因的腺相关病毒。[方法]设计靶向14-3-3ζ的siRNA,插入到psiRNA质粒H1启动子下游,Western-Blot检测siRNA的抑制效果。从psiRNA质粒上扩增H1-si-ζ基因表达单位亚克隆到AAV表达质粒AAV-MCS-EGFP的Mlu1酶切位点,构建重组质粒pAAV-14-3-3ζ-EGFP。重组质粒与包装质粒pAAV-RC和辅助质粒pHelper用磷酸钙法共转染AAV-293细胞制备重组病毒rAAV-14-3-3ζ-EGFP。重组病毒感染HT1080细胞,荧光显微镜检测病毒介导的EGFP表达。[结果]通过酶切鉴定和测序确定重组病毒载体pAAV-14-3-3ζ-EGFP已构建成功。病毒感染的细胞中检测到绿色荧光,表明重组病毒成功包装。[结论]成功包装出重组腺相关病毒AAV-14-3-3ζ-EGFP,为今后利用腺相关病毒载体进行14-3-3ζ相关的体内、外研究提供了试验基础。

[Objective] To construct the recombinant adeno-associated virus（AAV） vector containing siRNA targeting 14-3-3ζ.[Method] The siRNA was designed and synthesized,then inserted into plasmid psiRNA H1 promoter downstream.Western-Blot method was adopted to detect the inhibitory effects of siRNA.H1-si-ζ was amplified from psiRNA-14-3-3ζ,and then subcloned into plasmid pAAV-MCS-EGFP.Recombinant plasmid pAAV-14-3-3ζ-EGFP was co-transfected into AAV-293 cells with pAAV-RC and pHelper for recombinant AAV packaging.Expression of EGFP protein mediated by AAV was detected in the AAV-infected HT1080 cells under fluorescent microscope.[Result] The recombinant plasmid pAAV-14-3-3ζ-EGFP was verified by double digestion and sequence analysis.The detected green fluorescence indicated that recombinant AAV was successfully packaged and mediating gene expression.[Conclusion] The successfully packaged AAV-14-3-3ζ-EGFP provided references for further experiments of 14-3-3ζ analysis.