腺病毒伴随病毒表达载体表达人乳头状瘤病毒16型E6/E7基因的构建及应用

目的　为了了解人乳头状瘤病毒 (Humanpapillomavirus ,HPV) 1 6型的E6 /E7基因在细胞恶性转化中所起的作用 ,利用腺病毒伴随病毒载体 (AAVHelper -FreeSystem)构建和表达人乳头状瘤病毒 1 6型E6 /E7基因。方法　在pLXSN1 6E6E7质粒中经PCR扩增回收HPV 1 6E6E7基因片段 ,连接于T载体上进行测序 ,将正确的HPV 1 6E6E7插入pAAV -IRES -hrGFP质粒 ,协同pAAV -RC质粒和pHelper质粒共转染HEK 2 93细胞 ,包装表达HPV 1 6E6E7基因的重组腺病毒伴随病毒 ,收获病毒 ,并检测病毒的感染效率。结果　在包装细胞系HEK 2 93细胞中能形成较高感染效率的腺病毒伴随病毒 ,激光共聚焦检测可发现HEK 2 93细胞内有绿色荧光蛋白表达 ,HEK 2 93细胞经PCR可扩增出特异性的HPV 1 6E6E7基因片段 ,经流式细胞仪检测重组病毒的感染效率为71 3%。结论　携带人乳头状瘤病毒 1 6型E6E7基因的腺病毒伴随病毒可感染细胞 ,并在细胞内表达 ,可望用于宫颈癌病因学的研究

Construction of the expression plasmid pAAV-IRES-hrGFP encoding the human papillomavirus 16 E6E7

Objective To construct the expression plasmid pAAV-IRES-hrGFP encoding the human papillomavirus 16 E6E7 for study of the relationship between human papillomaviruse and cervical cancer.Methods The HPV 16 E6E7 DNA fragment was obtained by nested PCR from the pLXSN 16 E6E7,then ligated into pMD18-T vector and sequenced.The recombinant expression plasmid pAAV-IRES-hrGFP-E6E7 was constructed by inserting the E6E7 DNA fragment.pAAV-IRES-hrGFP-E6E7,pAAV-RC and pHelper was co-transfected into HEK 293 cells.The viral particles were harvested,and the infection efficiency was detected.Results The viral particles with high infection efficiency were generated in the packaging cell line HEK 293 cells.Green fluorescence protein was observed by laser confocal microscopy.The HPV16 E6E7 gene expression was confirmed by PCR.The infection efficiency was 71.3% by flow cytometry.Conclusions The recombinant expression plasmid pAAV-IRES-hrGFP-E6E7 may infect and be expressed in cells and the cells expressing HPV 16 E6E7 may be a useful tool for research on the etiology of cervical cancer.