南方医科大学学报2011，Vol.31Issue(4)：633-636,4.
鸭乙型肝炎病毒YMDD突变株体外耐药模型建立
Construction of a duck hepatitis B virus YMDD mutant and identification of its resistance phenotype
摘要
Abstract
Objective To construct a lamivudine-resistant plasmid containing 1.2 unit genome of duck hepatitis B virus and identify its replication and drug-resistance in avian LMH hepatica cells. Methods The recombinant plasmid PBS-DHBV1.2 was constructed using the 1.2-genome length DHBV DNA sequence from a dimer DHBV genome with pcDNA3.1 as the template. With site-directed mutagenesis, we obtained PBS-DHBV1.2-M512V plasmids with polymerase gene mutation from PBS-DHBV1.2. Two constructed plasmids were transiently transfected into LMH cells using FuGENETM6 transfection reagent and cultured in the medium containing different concentrations of lamivudine. Southern blot hybridization was performed to detect DHBV replication intermediates. Results PCR amplification, restriction digestion and plasmid sequencing all confirmed successful construction of PBS-DHBV1.2-M512V recombinant plasmid. Southern blot analysis identified the presence of all the expected DHBV replication intermediates in LMH cells. The replication capacity of the mutant plasmid was decreased by 2.7 times compared with that of the wild plasmid. The Icso of lamivudine was 37.12±8.81 ng/ml for the mutant, greater than that of the wild plasmid (10.90±4.80 ng/ml). Conclusion Compared with the wild plasmid, the mutant plasmid has a lower replication capacity and sensitivity to lamivudine in vitro.关键词
鸭乙型肝炎病毒/定点突变/鸡肝癌细胞系/拉米夫定Key words
duck hepatitis B virus/ site-directed mutagenesis/ LMH cells/ lamivudine分类
医药卫生引用本文复制引用
付喜花,梁蔚芳,吴小东,沈国俊,何海棠,陈金军,侯金林..鸭乙型肝炎病毒YMDD突变株体外耐药模型建立[J].南方医科大学学报,2011,31(4):633-636,4.基金项目
国家重点基础研究发展计划(973计划)(2007CBS 12901) （973计划）
