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

作　　者：付喜花[1] 梁蔚芳[1] 吴小东 沈国俊[1] 何海棠[1] 陈金军[1] 侯金林[1] 

机构地区：[1]南方医科大学南方医院感染内科,广东广州510515 [2]衡阳市中心医院消化内科,湖南衡阳421001

出　　处：《南方医科大学学报》2011年第4期633-636,共4页Journal of Southern Medical University

基　　金：国家重点基础研究发展计划(973计划)(2007CBS12901)~~

摘　　要：目的构建鸭乙型肝炎病毒(DHBV)YMDD点突变的复制质粒,研究其在鸡肝癌细胞中的复制及拉米夫定抗性。方法以广东樱桃谷鸭DHBV分离株为模板,将3631 bp的片段克隆至载体pBluescript II KS(+)中,构建含1.2倍病毒全基因的重组质粒,经PCR定点突变获得YMDD突变株。将构建质粒经FuGENETM 6瞬时转染鸡肝癌细胞,分析变异株的复制能力及拉米夫定耐药性。结果 PCR鉴定、酶切鉴定及序列测定均证实成功获得1.2倍DHBV全基因YMDD突变质粒。Southern印迹杂交表明重组质粒转染细胞后可检测到病毒复制中间体;野生株的复制能力是突变株的2.7倍。拉米夫定对变异株的IC50=37.12±8.81 ng/ml,高于野生株的IC50=10.90±4.80 ng/ml。结论与野生株相比,YMDD突变株质粒体外复制活性减弱,对拉米夫定敏感性下降。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 IC50 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 

分 类 号：R512.62[医药卫生—内科学]