乙型肝炎病毒拉米夫定耐药毒株的全基因质粒构建及其在HepG2细胞中复制与表达

目的 探讨拉米夫定耐药变异对病毒复制及抗原表达的影响.方法 通过定向点突变方法,成功的在模板质粒P3.8Ⅱ基础上构建成功3种P基因变异株质粒,并转染HepG2细胞,与HBV野毒株相比,分析变异毒株复制表达活性的差别.结果 经DNA测序证实,成功构建了rtG172E、rtG174C、rtG172E/rtG174C等3种变异株质粒,转染细胞系后发现上清均表达有HBsAg和HBeAg,证明转染质粒在细胞内成功表达并分泌HBsAg和HBeAg.通过检测细胞内复制产生的子代HBVDNA发现,rtG172E、rtG174C、rtG172E/rtG174C等3种变异株的复制活性均有明显减弱.结论 新发现的3种P基因变异株会影响HBV复制活性,对药物敏感性的影响需进一步研究.

Construction of whole-genome lamivudine-resistant hepatitis B virus mutant and its replication and expression in HepG2 cells

Objective To observe the changes of replication and antigen expressions of lamivudine-resistant hepatitis B virus(HBV) with polymerase gene mutation.Methods With site-directed mutagenesis,we constructed 3 HBV plasmids with polymerase gene mutation based on the template P3.8II.All the plasmids were transfected into HepG2 cells,in which the replication and expression of the virus were analyzed.Results The 3 polymerase gene mutant HBVs were successfully constructed.HBsAg and HBeAg expressions were detected in the supernatants of HepG2 cells transfected with these plasmids.The replication of the 3 mutant plasmids with rtG172E,rtG174C and rtG172E/rtG174C mutation decreased significantly in comparison with the wild-type virus.Conclusion The 3 polymerase gene mutant HBVs shows depressed capacity for viral replication,and in vitro drug sensitivity study is needed to establish the relationship between these mutations and nucleoside analogue resistance.