人乳头状瘤病毒癌蛋白E6和E7双标记质粒的构建及应用

Construction and Application of Human Papillomavirus-16 Oncoproteins E6 and E7 Plasmids with Double-Selection Markers

目的:构建人乳头状瘤病毒(human papillomavirus,HPV)-16 E6、E7癌蛋白及其突变型的双筛选标记质粒并筛选出稳定表达HPV-16癌蛋白的肺癌A549细胞株。方法:以携带新霉素抗性基因neo的pEGFP质粒(pEGFP-N1)为空载体,在EcoRⅠ和BamHⅠ位点间插入HPV-16 E6、E7及其突变型基因。新构建的质粒鉴定后转染A549细胞并用G418筛选,多次挑取单克隆后用流式细胞仪分选带荧光的细胞。结果:PCR、双酶切鉴定结果及DNA序列测定结果均证实质粒构建正确;PCR扩增结果显示细胞中存在目的基因;流式细胞术结果显示细胞阳性率高;Western blotting结果显示细胞能表达HPV-16 E6、HPV-16 E7蛋白。结论:成功构建pEGFP-E6、E7质粒并筛选出稳定表达E6和E7癌蛋白的A549细胞株,为进一步研究HPV对肺癌的影响奠定了基础;同时发现G418筛选结合流式细胞仪分选可提高稳定转染细胞的阳性率。

Objective： To construct plasmids with double-selection markers including human papillomavirus-16 E6 or E7 or their mutants and screen the stably transfected lung cancer A549 cells. Methods： pEGFP plasmid with neomycin resistance gene was used as empty vector and HPV-16 E6, E7 and their mutant genes were inserted between EcoR I and BamH I. The recombination ofpEGFP-16 E6, E6 mutant, E7 and E7 mutant plasmids were identified by PCR, restriction enzyme digestion, and sequencing. G418 was used to screen the recombinant plasmids-transfected A549 cells. The monoclones were picked out repeatedly and culture expansion. Fluorescence cells were sorted by flow cytometer. Results： The results of PCR and restriction enzyme digestion clearly appeared target bands. BLAST showed that the insert sequence was the same with the original one, and HPV-16 E6 and E7 sequences were the same with the homologous fragments of HPV-16 genome DNA. The target genes were amplified by PCR. Western blotting results showed that these cells could express HPV-16 E6 or E7 oncoprotein stably. Conclusions： pEGFP-HPV- 16 E6, E6 mutant, E7, and E7 mutant plasmids were constructed successfully and stably transfected cells were selected, which lay a foundation to study the influence of HPV on lung cancer. Additionally, the screening of G418 combined with the sorting of flow cytometry can increase the positive rate of transfected cells.