中文摘要：

目的构建HPV16 E7慢病毒表达载体。方法用RT-PCR方法从Caski细胞中扩增HPV16 E7基因的编码区序列,对扩增出目的片段酶切纯化后将目的片段交换连接入慢病毒表达质粒,酶切鉴定并测序正确后进行慢病毒包装和滴度测定。Western-blot验证3FLAG-E7融合蛋白在293 T细胞中的表达。结果成功获得HPV16 E7基因,成功将HPV16 E7克隆到慢病毒表达质粒中,且包装产生的慢病毒颗粒能成功表达3FLAG-E7融合蛋白。结论成功构建HVP16 E7和flag基因共表达的慢病毒表达载体,为进一步研究HPV16 E7基因的功能建立了高效稳定的转基因技术平台。

英文摘要：

To further study on the function of HPV16 E7 gene,we constructed a lentiviral expression vector carrying HPV16 E7.We amplified the full-length HPV16 E7 gene from the total RNA of the Caski cell by RT-PCR technique,then subcloned the gene into pLV-UbC-3FLAG vector,which was identified and confirmed by restricting enzyme digestion and DNA sequencing.After the above steps,recombinant lentiviruses were produced in 293T cells following the cotransfection of pLV-3FLAG-E7,packaging plasmids of pCD/NL-BH and pLTR-G.ELISA showed the titer of the virus was 2×108 TU/ml.Western blotting detected the expression of 3FLAG-E7 fusion protein in 293T cells.The results above indicate that the recombinant lentiviruse Lenti-E7 is successfully produced,which could be a foundation for our study the molecular function of HPV16 E7.