乙型肝炎病毒P基因反转录酶区I233V变异对阿德福韦酯耐药的实验研究

目的 构建含有HBV P基因反转录酶区I233V(rtI233V)突变的HBV复制型质粒,并分析此突变对阿德福韦(ADV)酯耐药性的影响.方法 利用定点突变技术构建带有rtA181V、rtI233V和rtN236T突变的HBV重组质粒,分别转染肝细胞系Huh7,加入含有不同浓度ADV的培养液,培养3 d后收集细胞DNA,用Southern印迹法检测HBV复制中间体.根据Southern印迹法得到的图像,用QUANTITIY ONE软件对其进行分析,应用Table Curve2D软件,通过公式拟合药物浓度-病毒复制率曲线.计算出ADV对各个重组质粒的半数抑制浓度(IC50)值后,以其对野生株质粒的IC50值为标准,分别计算各重组质粒的耐药倍数.结果 ADV对带有rtI233V突变的重组质粒的IC50值为(1.69±0.52)μmol/L,耐药倍数是野生株的6.5倍.结论 rtI233V变异能导致HBV对ADV轻度耐药.

Abstract：

Objective To construct an in vitro phenotypic analysis technology for evaluating the impact of I233V mutation in the hepatitis B virus(HBV)reverse transcriptase(rt)domain on adefovir (ADV)resistance.Methods A site-directed mutagenesis was used to construct recombinant HBV plasmid containing rtI233V,rtA181V and rtN236T.The culture solution with varied concentration ADV was added after the transient transfection of hepatocyte-derived cell lines with recombinant wild type and mutant HBV clones.Three days later,the cells were harvested and the replicable intermediate was detected by the Southern blot assay.The half maximal inhibitory concentration (IC50) and folds of resistance were calculated by the Table Curve2D software according to the Southern blot results.Results The variant harboring rtI233V mutation exhibited a 6.5-fold decrease of susceptibility to ADV with IC50 of(1.69±0.52)/μmol/L compared to the wild type HBV.Conclusion The findings suggest that the emergence of a single substitution at position rtI233V is sufficient to induce resistance to ADV.

Impact of I233V mutation in the hepatitis B virus reverse transcriptase domain on adefovir resistance

Objective To construct an in vitro phenotypic analysis technology for evaluating the impact of I233V mutation in the hepatitis B virus(HBV)reverse transcriptase(rt)domain on adefovir (ADV)resistance.Methods A site-directed mutagenesis was used to construct recombinant HBV plasmid containing rtI233V,rtA181V and rtN236T.The culture solution with varied concentration ADV was added after the transient transfection of hepatocyte-derived cell lines with recombinant wild type and mutant HBV clones.Three days later,the cells were harvested and the replicable intermediate was detected by the Southern blot assay.The half maximal inhibitory concentration (IC50) and folds of resistance were calculated by the Table Curve2D software according to the Southern blot results.Results The variant harboring rtI233V mutation exhibited a 6.5-fold decrease of susceptibility to ADV with IC50 of(1.69±0.52)/μmol/L compared to the wild type HBV.Conclusion The findings suggest that the emergence of a single substitution at position rtI233V is sufficient to induce resistance to ADV.