人乳头状瘤病毒16型E6基因短发夹结构RNA慢病毒载体的构建及鉴定

Construction and Identification of Short Hairpin RNA Lentiviral Vector of Human Papillomavirus(HPV) 16 E6

目的以人乳头瘤病毒(human papillomavirus,HPV)16型E6基因为靶点,构建小发夹状RNA(small hairpin RNA,shRNA)慢病毒载体.方法将HPV 16E6基因的特异性序列,应用基因重组技术插入到包含U6启动子及绿色荧光蛋白(GFP)报告基因的慢病毒载体pGCL-GFP中.重组质粒命名为pGCL-GFP-HPV16-E,测序鉴定后与慢病毒包装质粒pHelper 1.0及pHelper 2.0通过lipofectamine 2000共转染293T细胞,病毒液根据绿色荧光蛋白(GFP)表达水平测定滴度.结果 PCR扩增和测序结果证实HPV16 E6 shRNA核苷酸链序列插入正确,包装慢病毒产生病毒悬液,测定滴度为2×109 TU/mL.结论应用基因重组技术等方法,可成功构建HPV16 E6 shRNA慢病毒载体.

Objective To establish a lentiviral vector-mediated RNA interference（RNAi）of Human Papillomavirus（HPV）16 E6 gene.Methods Using genetic recombination technology,a pair of complementary small hairpin RNA（shRNA）oligonucleotides targeting the HPV16 E6 gene was cloned into lentiviral expression vector pGCL-GFP,which contained U6 promoter and green fluorescent protein reporter.The recombined plasmid named pGCL-GFP-vshHPV 16 E6 was confirmed by polymerase chain reaction and sequencing,and was cotransfected with pHel per 1.0 and pHel per 2.0 packaging plasmids into 293T cells by lipofectamine 2000;the lentivirus titer was determined according to the expression level of green fluorescent protein（GFP）.Results PCR and DNA sequencing demonstrated that the inserted sequences were correct.The titer of virus was 2E＋9 TU /ml.Conclusion A lentiviral shRNA expression vector targeting the HPV16 E6 gene is successfully constructed.