利用重叠延伸PCR快速构建中国株乙型肝炎病毒的感染性克隆

摘要

目的：体外扩增中国株乙型肝炎病毒(hepatitis B virus，HBV)的基因组全长序列，快速构建中国株HBV感染性克隆，建立中国株HBV体外复制细胞模型。方法：设计保守引物，扩增HBV全长基因组序列。对扩增到的HBV基因组进行DNA测序及计算机辅助分析，确定病毒的基因型。通过重叠延伸PCR(splicing by overlap extension PCR，SOE-PCR)技术，构建1.3倍基因组长度的HBV感染性克隆质粒pHBV1.3。将pHBV1.3质粒转染人肝癌细胞系HepG2，采用Western Blot、酶联免疫吸附试验及Real-PCR检测病毒复制及表达情况。检测该感染性克隆对临床抗病毒药物阿德福韦的敏感性。结果：成功扩增到1株C基因型HBV全长基因组，其GenBank登陆号为KF495606。构建了中国株HBV感染性克隆pHBV1.3(C)质粒，该质粒能在肝癌细胞株中进行有效的复制、转录和表达。阿德福韦能在体外抑制该HBV感染性克隆的复制，抑制程度依赖于药物浓度。结论：利用SOE-PCR可快速构建HBV感染性克隆，所构建的感染性克隆能介导高水平病毒复制，可用于HBV复制机制和抗病毒等方面的研究。%Objective: To clone the full-length genome of hepatitis B virus(HBV) (China isolate), construct HBV infectious replicon, and consequently establish cell model for HBV replication in vitro. Methods: Using conserved primers, the full-length genome of HBV was cloned from clinical serum sample. Then, 1.3-fold overlength genome of HBV, from the obtained HBV genome, was constructed by splicing of overlap extension PCR(SOE-PCR) and then cloned into pBluescript KS(+) to generate recombinant plasmid of pHBV1.3. The recombinant plasmids of pHBV1.3 were further transfected into HepG2 cell line. ELISA were performed to detect the levels of HBsAg and HBeAg in supernatant of HepG2 cells, Western Blot was utilized to reveal the level of intracellular HBsAg and HBeAg, and real-time PCR was used to analyze the titer of HBV DNA in supernatant. Antiviral effect of adefovir was evaluated in pHBV1.3(C) transfected HepG2 cells. Results: The full-length genome of HBV (China isolate) was successfully cloned from clinical serum sample, and DNA sequencing result revealed that the genotype of the obtained HBV(GenBank accession No. KF495606) belongs to C type. The recombinant plasmid of HBV infectious replicon was constructed from cloned HBV genome and named pHBV1.3(C). HBV gene carried in pHBV1.3(C) could efficiently replicate and express in HepG2 cells. Adefovir could inhibit HBV replication in this HBV cell model and the inhibition effect depended on the concentration of drug. Conclusion: The infectious replicon of HBV could be rapidly constructed by SOE-PCR, the obtained HBV construct could initiate viral replication efficiently in pHBV1.3(C) transfected hepatoma cells, which provide a useful in vitro cell model for HBV research.