HBV临床分离株复制型质粒的构建与鉴定

Construction and identification of plasmids containing a replication-competent HBV supergenomic DNA derived from patients with chronic HBV infection

目的克隆HBV临床分离株全长基因组,构建真核表达复制载体,为研究HBV的耐药机制提供基础。方法从乙型肝炎患者血清中提取HBV DNA,PCR扩增HBV全长基因组,经SapI酶切后,克隆入pHY106载体,测序分析序列,然后用lipofectamine 2000将重组载体转染Huh7细胞,3 d后收集培养上清液,ELISA检测HBsAg、HBeAg水平;Real-time PCR检测细胞培养上清液中HBV含量;Southern印迹检测细胞内HBV复制中间体。结果获得5株HBV临床分离株均为野生型,其中基因B型3株,基因C型2株。成功构建5株HBV临床分离株的复制型质粒,在转染Huh7细胞的上清液中检测到HBsAg、HBeAg及HBV DNA,细胞内检测到HBV复制中间体。结论成功克隆并构建了HBV临床分离株复制型质粒,这种复制型质粒将在HBV临床分离株的耐药机制和药物敏感性等方面研究中发挥重要作用。

Objective To amplify full length hepatitis B virus（HBV）genome derived from patients with chronic HBV infection,and construct plasmids containing a replication-competent HBV supergenomic DNA.Methods Full-length HBV genomes were purified from serum of chronic hepatitis B（CHB）patients,and amplified by high fidelity polymerase chain reaction（PCR）,The PCR products were then restricted with Sap I and ligated with Sap I-linerized pHY106 plasmid,constructs of which were sequenced and transfected into Huh7 cells.Enzyme-linked immunosorbent assay,real-time PCR,and Southern blot,were performent to investigate the HBV gene expression and replication respectively.Results Five HBV wild type isolates（3 of genotype B and 2 of genotype C）,denived from CHB patients,were amplified successfully,corresponding replication competent plasmids of which were constructed.In the recombinants-transfected Huh7 cells,high HBV DNA load,and high levels of HBsAg,and HBeAg were detected in the supernatants,and HBV core associated replicative intermediates were verified.Conclusion The construction of replication competent plasmids for HBV isolates from CHB patients will play an important roles in the detection and mechanism research for HBV mutation,as well as salvage therapy for patient infected with nucleos（t）ide analogues-associated mutants.