一种基于人miR-30结构高效干扰LMP1基因表达的慢病毒载体的构建

Construction and characterization of the lentiviral vector carrying a miR- 30a- based shRNA targeting EBV LMP1 oncogene

目的:构建慢病毒载体并利用miRNA原理进行LMP1病毒癌基因干扰的可行性及有效性研究。方法:基于人miR-30a结构,利用靶点分析软件设计4个LMP1的候选干扰靶点,报告基因EGFP与miRLMP1靶点形成共顺反子表达,靶点载体质粒与LMP1高表达质粒用脂质体共转染工具细胞293FT,用Western blot方法筛选最有效的干扰靶点并用三质粒系统在293FT细胞中包装慢病毒,进行滴度鉴定。结果:在293FT工具细胞中成功筛选到一个高效干扰LMP1基因的靶点序列,在Western blot水平的蛋白干扰率高达95%;在20ml培养上清中可获得具有活性慢病毒颗粒7.2×108个,经纯化浓缩获得重组慢病毒颗粒,滴度高达6×109TU/ml,经流式细胞仪检测在80MOI时其感染EBV阳性的鼻咽癌细胞株C666-1的感染率高达80%以上。结论:基于人miR-30a结构的miR-LMP1能高效干扰LMP1基因的蛋白表达,利用慢病毒载体来建立稳定基因干扰肿瘤细胞株可能是一个良好的方法。

To investigate the feasibility and validity of the construction of a lentiviral vector carrying shRNA against EBV LMP1 oncogene. Methods：Based on miR - 30a sequence,four LMP1 - targeted RNAi shRNA candidate sequences were designed and cloned alone with EGFR reporter gene as a cocistron. The plasmid carrying LMP1 shRNA and the LMP1 - expressing plasmid were cotransfected into 239FT cells,and screened the most effective shRNA targeting LMP1 and constructed lentiviral vector and characterized. Results：The most effective shRNA silen-cing viral LMP1 oncogene was screened successfully and more than 95% LMP1 protein was knocked down using Western blotting analysis. In 20ml 239FT cell culture supernatant,about 7. 2 × 10^8 viable particles of recombinant lentivirus was packaged and the titer was as high as 6 × 10^9 TU/ ml after purification and condensation. More than 80% of EBV - positive NPC cells C666 - 1 were infected at 80MOI through flow cytometry detection. Conclusion：A miR - 30a - based shRNA targeting EBV LMP1 oncogene can knock down effectively viral LMP1 expression and lentiviral vector may be a substitutive selection for establishment of a stable RNAi NPC cell line.