慢病毒法建立稳定表达HPV-16 E6癌蛋白的肺癌细胞株  

Establishment of lung cancer cell lines with stable HPV-16 E6 expression using lentiviral method

作　　者：冯芸[1] 丰晓威 刘锦华[1] 刘飞[1] 李祥勇[1] 兰柳波[1] 陈小谊[1] 唐旭东[1] 

机构地区：[1]广东医学院生物化学与分子生物学研究所,广东湛江524023

出　　处：《广东医学院学报》2014年第2期129-133,共5页Journal of Guangdong Medical College

基　　金：国家自然科学基金项目(No.81372511)

摘　　要：目的方法结果结论(HPV)-16 E6DNA HPV-16 E6(PLIG)(PLIG-E6)293T NCIH460 G418 4 DNA PCR Western blotting NCIH460 HPV-16 E6 DNA NCI-H460 HPV-16 E6 DNA HPV-16 E6探讨慢病毒法建立人乳头瘤病毒癌蛋白表达肺癌细胞株的可行性。采用重组技术将基因插入到含有增强型绿色荧光蛋白基因的慢病毒质粒载体中,获得重组慢病毒载体,经双酶切和测序鉴定正确后,与慢病毒包装载体一起转染细胞,收集病毒液,测定滴度后,感染肺癌细胞株。经筛选培养周后,提取细胞基因组和蛋白,分别用和方法分析细胞中和蛋白的表达。双酶切及测序结果表明重组慢病毒表达载体构建成功;感染慢病毒液的细胞中可检测出和蛋白表达。用慢病毒法成功构建稳定高表达癌蛋白的肺癌细胞株。Objective To study the feasibility of the construction of lung cancer cell lines with human papillomavirus（HPV）-16 E6 expression using lentiviral method. Methods Using DNA recombinant technique, the recombinant vector PILGE6 was acquired by insertion of HPV-16 E6 gene into lentiviral transfer plasmid PLIG containing enhanced green fluorescent protein. After identified by double digestion and DNA sequencing, the recombinant plasmid was packaged with packaging vectors and transfected with 293 T cells. The lung cancer cell line NCI-H460 was transfected with recombinant vector PILG-E6 after lentivirus titer was determined. The transfected cells were cultured with G418 media for 4 weeks, followed by detection of HPV-16 E6 DNA and protein using PCR and Western blotting, respectively. Results Double digestion and DNA sequencing revealed the successful construction of the recombinant lentiviral expression vector. HPV-16 E6 DNA and protein were expressed in lentivirus-infected NCI-H460 cells. Conclusion Lung cancer cell line with stable overexpression of HPV-16 E6 oncoprotein can be successfully constructed by lentiviral method.

关 键 词：HPV-16 E6 慢病毒方法 肺癌 质粒 

分 类 号：Q78[生物学—分子生物学] R73[医药卫生—肿瘤]