携带人肝癌细胞CDK2基因的rAAV载体的构建与鉴定

Construction and identification of rAAV vector carrying the CDK2 gene in human liver cancer cells

目的构建携带人肝癌细胞CDK2基因的重组腺相关病毒(rAAV)载体;方法设计能表达CDK2特异性的小干扰RNA(siRNA),构建pAKD-shRNA-CDK2重组质粒,采用磷酸钙共转染法将重组质粒pAKD-shRNACDK2、包装质粒pAAV-RC和辅助质粒pHelper共转染AAV-293细胞,采用定量PCR方法对病毒进行滴度测定;结果三质粒共转染AAV-293细胞48h后,荧光显微镜下检测到GFP绿色荧光,表明共转染成功,测定病毒平均滴度为3.56×1012v.g/ml。结论成功构建携带人肝癌细胞CDK2基因的重组腺相关病毒rAAV-shRNA-CDK2。

Objective CDK2 gene construct carrying human hepatoma cell recombinant adeno-associated virus(rAAV)vector.Methods According to human liver cancer cell CDK2 gene sequence,to design of specific shRNA sequences against CDK2.Construct recombinant plasmids pAKD-shRNA-CDK2,the recombinant plasmid pAKD-shRNACDK2,packaging plasmid pAAV-RC and helper plasmids pHelper were co-transfected AAV-293 cells using the calcium phosphate method.The titer was measured by Real-time PCR.Results Three plasmids were co-transfected AAV-293 cells after 48 h,GFP green fluorescence was detected by fluorescence microscopy,indicating the success of co-transfected;Determination of titer of packaging success rAAV-shRNA-CDK2 was 3.56×1012 v.g./ml.Conclusion The experiment was successfully constructed with the purpose of CDK2 gene recombinant adeno-associated virus rAAV-shRNACDK2.