中文摘要：

目的分别构建HPV16 E5、E6和E7癌基因的重组慢病毒载体感染人口腔上皮细胞,为研究E5基因对口腔上皮细胞的致癌机制奠定基础。方法克隆HPV16 E5、E6和E7基因,分别构建pLVX-Ac GFP-E5、pLVX-Ac GFP-E6和pLVX-Ac GFP-E7重组慢病毒载体,与包装质粒共转染293T细胞。收集3种慢病毒上清液,分别感染口腔上皮细胞、腺嘌呤素筛选感染细胞,RT-PCR和Western blot法检测目的基因的表达。结果成功构建pLVX-Ac GFP-E5、pLVX-Ac GFPE6和pLVX-Ac GFP-E7重组慢病毒载体,获得3种慢病毒上清液,筛选得到3个稳定的细胞株。RT-PCR和Western blot均可检测到HPV16 E5、E6和E7基因在口腔上皮细胞内表达。结论本研究构建的3个重组慢病毒载体能成功感染口腔上皮细胞。

英文摘要：

Objective To provide the basis for the study of HPV16 E5 gene inthe pathogenesis of oral squamous cell carcinoma by constructing three lentiviral vectors expressing HPV16 E5,E6 or E7 oncogene and transfecting oral epithelial cells with the vectors. Methods HPV16 E5,E6 or E7 oncogene was amplified using PCR and ligated into the lentiviral vector p LVX-Ac GFP-N1 respectively to construct vectors p LVX-Ac GFP-E5,p LVX-Ac GFP-E6 and p LVX-Ac GFP-E7,then respectively cotransfected 293 T cells with packaging plasmids,viral supernatant was collected to respectively transfect oral epithelial cells. Afterpuromycin screening,oral epithelial cells with HPV 16E5,E6,or E7 oncogene transfection were constructed,then reverse transcription PCR and western blotassays were performed for verifying HPV16 E5,E6 or E7 expression. Results p LVX-Ac GFP-E5,p LVX-Ac GFP-E6 and p LVX-Ac GFP-E7 were successfully constructed,oral epithelial cells expressing HPV 16 E5,E6 or E7 oncogene wereacquired,HPV16 E5,E6 or E7 expression was confirmed in oral epithelial cellsthrough reverse transcription PCR and western blot assays. Conclusion Three lentiviral vectors expressing HPV16 E5,E6 or E7 oncogene can successfully infect oral epithelial cells.