人SR-BI基因表达抑制慢病毒载体构建及其稳定细胞株的筛选  

Construction of recombinant lentivirus and screening of stable cell line with human SR-BI gene knock down

作　　者：高伟[1] 高荣[1] 任浩[1] 

机构地区：[1]第二军医大学微生物学教研室上海市医学生物防护重点实验室,上海200433

出　　处：《中国热带医学》2016年第3期203-207,共5页China Tropical Medicine

基　　金：国家自然科学基金(No.31370196);973计划(No.2013CB531601);军队基金(No.BWS14J023;2012JS01)

摘　　要：目的建立SR-BI表达抑制的肝癌细胞系,以作为研究SR-BI基因突变对HCV感染性影响的细胞模型。方法构建SR-BI短发夹状RNA(Short hairpin RNA,sh RNA)慢病毒重组质粒Lv-SR-BI-sh RNA,挑取克隆进行PCR扩增和序列测定;将阳性重组质粒与慢病毒辅助质粒共转染HEK2973T细胞,包装SR-BI sh RNA慢病毒并进行病毒滴度测定。SR-BI sh RNA慢病毒感染人肝癌细胞株Huh7和Huh7.5.1细胞,嘌呤霉素筛选,Real-time PCR和Western blot法检测SR-BI m RNA转录和蛋白表达。结果测序结果证实Lv-SR-BI-sh RNA慢病毒载体构建成功并获得HEK293T细胞中包装的慢病毒,Lv-SR-BI-sh RNA重组慢病毒感染Huh7和Huh7.5.1细胞,感染组SR-BI m RNA转录降低,蛋白表达降低。结论建立了SR-BI表达抑制的人肝癌细胞株Huh7-si SR-BI和Huh7.5.1-si SR-BI稳定细胞系。Objective To establish hepatocellular carcinoma cell line with human SR-BI knock down, which could beused as cell model to study the effect of SR-BI gene mutation on infectivity of hepatitis C virus. Methods SR-BI sh RNAwas designed and cloned into p GP-Lenti3 vector. Positive recombinant Lv-SR-BI-sh RNA was selected and verified by PCRand sequencing. Lv-SR-BI-sh RNA and helper plasmids were co-transfected into HEK293 T cells. The recombinant lenti-SR-BI-sh RNA virus was harvested, tittered, and used to infect hepatocellular carcinoma cell lines Huh7 and Huh7.5.1. Puromycinwas added into the culture medium to select for stably-transduced cells. Real-time PCR and Western blot were carried out todetect transcription and expression of SR-BI. Results Sequence verified that SR-BI sh RNA was successfully inserted intop GP-Lenti3 vector, and lenti-SR-BI-sh RNA viruses were obtained by co-transfection of Lv-SR-BI-sh RNA with helperplasmids into HEK293 T cells. Reduced transcription and expression of SR- BI was observed in Huh7 and Huh7.5.1 cellsinfected with lenti-SR-BI-sh RNA viruses. Conclusion Hepatocellular carcinoma cell lines Huh7-si SR-BI and Huh7.5.1-si SR-BI with human SR-BI knock down were successfully developed.

关 键 词：人SR-BI 短发夹状RNA 肝癌细胞系 构建 筛选 

分 类 号：R378.21[医药卫生—病原生物学]