耐拉米夫定乙肝病毒变异株的人工构建及其限制性片段长度多态性分析

Construction of HBV lamivudine-resistant mutant and the restriction fragment length polymorphism analysis

目的 建立简便易行的筛检常见的耐拉米夫定乙肝病毒变异株的方法。方法通过PCR定向点突变 ,构建最常见的耐拉米夫定乙肝病毒变异株 (YMDD变异株 ) ,并建立相应的错配PCR结合限制性片段长度多态性分析 (RFLP)检测此变异株。结果PCR成功引入点突变 ,构建了YMDD变异株的克隆 ,同时错配PCR结合限制性片段长度多态性分析可有效区分YMDD变异株和HBV野毒株。结论采用PCR的方法可诱导定向点突变 ,错配PCR结合限制性片段长度多态性分析可用于筛检临床常见的耐拉米夫定乙肝病毒变异株 (YMDD变异株 ) ,为临床监测提供了一种很好的模式。

Objective To establish an easy method for the detection of HBV lamivudine-resistant mutants Methods HBV YMDD mutant was constructed by site-directed mutagenesis A novel method of mismatched PCR in combination with enzyme digestion was used to distinguish the YMDD mutant from HBV wild type Results The HBV YMDD mutant was proved by sequencing By using the new method of PCR restriction flagment length polymorphism (RFLP), HBV wild type can be easily distinguished from HBV YMDD mutants Conclusion The novel PCR-RFLP can be applied to monitor HBV lamivudine-resistant mutants