HBV全基因组克隆转染细胞系的建立
作者：赵艳芳，闫永平，苏海霞，王安辉，张磊，张景霞，门可，徐德忠【关键词】 肝炎病毒,乙型；基因组,病毒；克隆,分子；转染；细胞培养；基因表达
【Abstract】 AIM: To construct a new cell culture system for producing HBV in vitro.METHODS: Fulllength HBV DNA was cloned into pcDNA3 vector (named pcDNA33HBV). HepG2 cells were transfected with pcDNA33HBV and screened with antibiotic G418. HBsAg and HBeAg were identified by ELISA and HBcAg was identified by immunocytochemical staining. S gene mRNA expression was tested by RTPCR and HBV DNA in the supernatant of transfected cells was tested by PCR. RESULTS: The plasmid pcDNA33HBV was constructed successfully. After stable transfection, HBsAg and HBeAg could be detected in the supernatant of transfected cells. HBcAg positive staining was located mainly in the cytoplasm. S gene mRNA expression was verified. S gene and preS gene could be detected in the supernatant by RTPCR. The copies of HBV genome can reach 1×108 copies/L detected by realtime quantitative PCR. CONCLUSION: Recombinant plasmid pcDNA33HBV could be expressed, transcribed and replicated in HepG2 cells. This transfectionbased cell culture system could produce high copies of HBV genome.
【Keywords】 hepatitis B virus; genome, viral; cloning, moleculor; transfection; cell culture; gene expression
【摘要】 目的： 建立HBV体外细胞培养体系. 方法： 构建HBV全基因克隆质粒pcDNA33HBV，稳定转染HepG2细胞，G418筛选. ELISA检测细胞上清液HBsAg，HBeAg的表达，免疫组化检测细胞内HBcAg的表达，RTPCR检测细胞S基因mRNA的表达，PCR检测细胞上清液DNA. 结果： 成功构建了HBV全基因克隆质粒pcDNA33HBV，稳定转染HepG2后，培养上清HBsAg，HBeAg阳性，细胞内HBcAg呈核浆型分布，且以浆型分布为主. RTPCR证实有HBV S基因 mRNA 的表达，上清中HBV S及前S基因阳性，荧光定量PCR检测显示培养上清中HBV 滴度达1×108拷贝/L. 结论： 重组质粒pcDNA33HBV能在HepG2细胞中表达、转录、复制，该细胞培养体系能产生较高水平的HBV.
【关键词】 肝炎病毒，乙型；基因组，病毒；克隆，分子；转染；细胞培养；基因表达
0引言
由于HBV宿主范围窄、动物模型缺乏，体外组织培养也一直没有太大进展，且不能在体外人工培养，制约了对HBV的研究［1］. 将HBV DNA转移至靶细胞，建立表达HBV的体外培养细胞模型对研究HBV生物学特性和肝炎发病机制有重要意义［2-4］. HBV基因组呈双链闭合环状，基因结构紧凑，其开放读框分布于全长DNA，为使完整的转染基因能在细胞内复制，目的'基因的长度要大于一个HBV DNA单元［5-6］. 我们构建3倍HBV基因的重组真核表达质粒，转染细胞，以筛选获得稳定表达病毒蛋白的细胞模型，并研究其产生HBV的水平.
1材料和方法
1.1材料HBV全基因亚克隆载体pUC193HBV为pUC19在EcoRⅠ及HindⅢ位点中插入头尾相连的HBV（adr亚型）三连体，由山东大学医学院免疫学研究所张秋博士惠赠，保存在大肠杆菌DH5α中. 真核细胞表达载体pcDNA3由第四军医大学生物化学与分子生物学教研室赵晶博士惠赠；人肝母细胞瘤细胞系（HepG2）由第四军医大学微生物学教研室惠赠，本室传代培养；内切酶EcoRⅠ, HindⅢ, T4 DNA连接酶,Taq DNA聚合酶,反转录酶及DNA marker（TakaRa公司）；质粒提取试剂盒（西安市莱博科技发展有限公司）；LipofectamineTM 2000（Invitrogen公司）；DMEM培养基，G418（Gibco公司）；新生小牛血清（杭州四季青生物制品有限公司）；ELISA检测试剂盒（上海科华