PIK3CA基因RNA干扰慢病毒载体的构建和鉴定

目的构建靶向PIK3CA（P110α）基因的RNA干扰慢病毒载体。方法针对PIK3CA—mRNA（NM_006218）设计4个RNA干扰靶点序列和一个阴性对照靶点，构建慢病毒转移质粒pGCL—GFP。构建成功的RNA干扰质粒与PIK3CA表达质粒共转染293T细胞，荧光显微镜下观测转染效果，Western Blot检测PIK3CA蛋白表达，筛选出干扰效果最好的RNA干扰质粒。将该质粒与两种辅助包装原件载体质粒共转染293T细胞，包装成慢病毒并检测其滴度。结果4个靶点和阴性对照靶点的RNA干扰质粒均构建成功；通过Westem Blot检测获得最有效干扰靶点，并成功包装成滴度为2×10^8TU／ml的慢病毒。结论成功构建可供感染的PIK3CA基因RNA干扰慢病毒载体，为进一步研究PIK3CA功能和用慢病毒进行卵巢癌基因治疗奠定了基础。

Construction of RNA interfering lentivirus vector targeting PIK3CA gene and its identification

Objective To construct the RNA interfering（RNAi） lentivirus vector targeting PIK3CA（P110 α）. Methods Four specific target sequences and a negative control sequence from PIK3CA-mRNA sequence（NM_006218） were designed to construct pGCL-GFP. 293T cells were co-transfected with the successfully constructed RNAi plamid and PIK3CA expression plamid. The transfection efficiency was observed under a fluorescence microscope. Western blot was used to detect the expression of PIK3CA protein. RNAi plasmid with an optimal interfering efficacy was screened and 293T cells were co-transfected with auxiliary packaging vectors to pack lentivirus and detect its titer. Results The RNAi plamid with four targets and a negative control sequence was successfully constructed. Western blot analysis showed that the interfering targets were optimal. The lentivirus with a titer of 2×10^8TU/ml was successfully packed. Conclusion RNA interfering lentivirus vector targeting PIK3CA gene can be constructed, which provides a foundation for investigating PIK3CA function and gene therapy for ovarian cancer using lentivirus.