一种构建HBV聚合酶特定位点多种突变的方法  

A method for constructing extensive mutagenesis of HBV polymerase

作　　者：刘亚[1] 杨柳青[1] 张文露[1] 黄爱龙[1] 胡接力[1] 

机构地区：[1]重庆医科大学感染性疾病分子生物学教育部重点实验室,重庆400016

出　　处：《重庆医科大学学报》2017年第7期771-775,共5页Journal of Chongqing Medical University

摘　　要：核苷(酸)类似物[neocleot(s)ide analogues,NUCs]在治疗乙型肝炎病毒(hepatitis B virus,HBV)感染的过程中常见的一个问题就是耐药的产生。耐药的产生与HBV逆转录酶(reverse transcriptase,RT)区特定位点突变具有直接关系。现有的体外表型分析方法存在固有的限制。本文提出一种构建HBV聚合酶特定位点多种突变的新方法,即位点特异性随机突变,通过一轮聚合酶链式反应扩增,在RT区特定位点引入多种突变形式,再结合golden-gate克隆法以及片段替换反应(fragment substitution reaction,FSR)将包含不同突变的DNA片段克隆到相应载体中以进行下一步分析。通过这种方法,构建了阿德福韦(adefovir,ADV)耐药位点rt181和rt236位点的多种突变形式。A common problem during long term administration of chronic hepatitis B with Neocleot （s）ide analogues （NUCs） is drug resistance. NUCs resistance has been attributed to mutations at specific sites in the reverse transcriptase domain of HBV polymerase. Currently used extensive mutagenesis assay based on plasmid transfection method has some disadvantages. Here, we developed an al- ternative method for extensive mutagenesis phenotype assay of HBV polymerase. This method combined a randomized mutation method and golden-gate or fragment substitution reaction （FSR） strategy developed previously. Using this way,we successfully constructed multiple mutations at rtl 81 and rt236, the well-known mutation sites related to ADV resistance.

关 键 词：克隆 生物工程方法 核苷类似物 耐药 HBV聚合酶 

分 类 号：R51[医药卫生—内科学]