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

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

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。

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.