乙型肝炎病毒前C区和BCP区突变株复制质粒的构建

Construction of recombinant replication competent plasmids containing HBV genome with mutations of precore and basic core promoter

目的 构建乙型肝炎病毒 (HBV)前C区和基本核心启动子区 (BCP)突变株的复制质粒。方法 以含 1 2倍拷贝HBVDNA全基因的质粒 (pHBV1 2 )为工具 ,采用分子克隆、PCR定点诱变、限制性酶切长度多态性和序列分析等技术构建目的质粒。转染肝癌细胞株Huh7后 ,测定培养上清HBsAg、HBVDNA来了解病毒抗原的表达和病毒复制。结果 成功构建了 10种HBV全基因前C区 /BCP区突变的表达质粒 ,转染肝癌细胞株 ,获得病毒的表达和病毒颗粒的分泌。其中 ,pUC HBVT176 2、A176 4双突变以及pUC HBVT175 3突变株复制效率较高 ,转染细胞培养上清的HBVDNA为3 16× 10 5拷贝 ml。野生型复制子培养上清HBVDNA含量稍低于BCP突变复制子。结论 获得 10株含有不同前C区和BCP区突变的HBV全基因复制质粒 ,为体外进一步研究上述变异的生物学意义提供基本模型。

Objective To construct a series of recombinant replication competent plasmids of hepatitis B virus (HBV) full-length genome with varying mutations of precore and basic core promoter Methods The plasmid containing the entire HBV (12 copies) genome was used to construct objective plasmids The objective competent vectors were constructed by molecular cloning and PCR-based site-directed mutagenesis in vitro and confirmed with sequence analysis After introducing the plasmids into hepatocellular carcinoma cell line (Huh7) by calcium phosphate transfection, the culture supernatant was collected to detect HBsAg and HBV DNA to analyze viral replication and expression Results Ten competent vectors with varying precore and basic core promoter mutations were constructed After transfecting into Huh7, the vectors replicated and secreted viral particles Conclusion Ten full-length competent HBV recombinants were obtained, which were harbouring varying mutations within precore and basic core promoter