【摘要】 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。更多还原

【Abstract】 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 EcoRⅠ and BamHⅠ.The recombination of pEGFP-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.更多还原