慢病毒介导稳定表达HBcAg的P815/c细胞株的建立及鉴定  

Establishment and Identification of P815/c Cell Line Stably Expressing HBcAg Mediated by Lentivirus

作　　者：戴胜兰[1] 卓萌[2] 汤正好[2] 臧国庆[2] 

机构地区：[1]江苏大学附属人民医院消化内科,镇江212000 [2]上海交通大学附属第六人民医院感染病科,上海200233

出　　处：《科学技术与工程》2017年第6期147-151,共5页Science Technology and Engineering

基　　金：国家自然科学基金面上项目(81270502)资助

摘　　要：研究旨在建立稳定表达HBcAg的P815/c細胞株,为评价针对HBcAg的乙肝疫苗的免疫效果提供体外感染的细胞模型。通过构建携带HBcAg基因的慢病毒表达载体质粒pLenti-Ubc-HBcAg-EGFP-3FLAG4RES-Puro,载体质粒携帯有加强型绿色荧光蛋白(eGFP)及嘌呤霉素(Puromycin)抗性标记基因,与包装质粒pLP1、pLP2以及包膜质粒pLP/VSVG共转染人胚肾293T细胞进行包装,得到携帯有HBcAg基因的重组慢病毒颗粒LV-HBcAg-Puro。经纯化、鉴定后转染小鼠肥大瘤细胞株P815细胞72h后,通过加入并维持2μg/mL的嘌呤霉素杀死未被有效感染的细胞,药物筛选约10 d后,免疫荧光及流式细胞仪检测表达GFP的细胞比例在98%以上,Western Blot可检测到转染后细胞内HBcAg的蛋白表达。成功构建稳定表达HBcAg基因的细胞株P815/C,为研究小鼠体内针对HBcAg的疫苗诱发的CTL反应提供了细胞杀伤实验的靶细胞。To establish the stable P815/c cell line expressing HBcAg, and to provide the cell infection model for evalution of the effect of the hepatitis B vaccine against HBcAg.The recombinant HBcAg lentiviral expression vector plasmid pLenti-Ubc-HBcAg-EGFP-3FLAG-IRES-Puro was constructed, which encoded HBcAg gene with enhanced green fluorescent protein （eGFP） gene and puromycin resistance marker gene.The recombinant lentivirus particles LV-HBcAg-Puro were generated by co-transfecting 293T cells with pLenti-Ubc-HBcAg-EGFP-3FLAG-IRES-Puro and the packaging plasmids pLP1, pLP2 and the envelope plasmid pLP/VSVG.Then, LV-HBcAg-Puro was purified and identified.72 h after transfection of P815 cells with LV-HBcAg-Puro, 2μg/mL puromycin was added and ineffective infected cells were killed.10 days after drug screening, the percentage of expressing GFP cells was over 98% detected by Immunofluorescence and flow cytometry.The expression of HBcAg was confirmed by western blot.The stable expressing of HBcAg gene cell line P815/c was established, providing the target cell for cytotoxic T cell response induced by vaccines against HBcAg in mice.

关 键 词：慢病毒 乙型肝炎核心抗原 P815细胞株 

分 类 号：R512.62[医药卫生—内科学]