三种乙型肝炎病毒拉米夫定耐药突变的酶切分析方法及其应用

Analysis of three lamivudine-resistant HBV mutants with the method of restriction enzyme digestion and its application

目的 建立三种乙型肝炎 (乙肝 )病毒拉米夫定耐药突变的酶切分析方法 ,了解这三种耐药突变在拉米夫定治疗患者中的发生情况。方法 设计 3对HBVP基因引物进行聚合酶链反应(PCR)扩增 ,应用NdeⅠ和NlaⅢ两种限制性内切酶对PCR产物进行酶切 ,酶切产物用电泳加以分析。应用此方法检测 70例拉米夫定治疗的慢性乙肝患者中 3种拉米夫定耐药突变的发生情况。结果 NdeⅠ和NlaⅢ两种限制性内切酶对PCR产物进行酶切可有效区分HBV野株和YMDD(酪氨酸 蛋氨酸 天冬氨酸 天冬氨酸 )变异株 ,并确定是YI(异亮氨酸 )DD或YV(缬氨酸 )DD变异。用NlaⅢ内切酶对PCR扩增产物酶切可有效区分HBV野株和L5 2 6M(第 5 2 6位氨基酸由亮氨酸变为蛋氨酸 )变异株。利用此方法发现 11例患者在服用拉米夫定过程中出现耐药突变 ,其中YIDD变异 6例 ,YVDD变异 5例 ,后者有 1例伴有L5 2 6M点突变。结论 本方法只需应用常规PCR和酶切技术 ,既可有效筛检出YMDD变异株标本 ,还可以确定属于YIDD还是YVDD变异 ,或是否伴有L5 2 6M点突变等 ,均可作为临床监测拉米夫定耐药性的参考。

Objective To investigate the prevalence of three lamivudine resistant HBV mutants in lamivudine treated Chinese patients. Methods Using three pairs of HBV polymerase gene primers,HBV polymerase gene B and C domain fragments were amplified by PCR.The PCR products were then digested by restriction enzyme Nde Ⅰ and Nla Ⅲ.The digested products were analyzed by electrophoresis.With this method,the prevalence of the three lamivudine resistant mutants in lamivudine treated Chinese patients was investigated.Results After Nde Ⅰ digestion of p24 and p29 amplified product,HBV wild type could be easily separated from YMDD mutant.At the same time,YIDD could be separated from YVDD mutant after Nla Ⅲ digeston of p24 and p29 amplified product.HBV wild type could be separated from L526M mutant after Nla Ⅲ digestion of p23 and p29 amplified product.By this method,the authors found that these eleven patients were infected with lamivudine resistant mutants.Six of them were infected with M5501 mutant;five were infected with M550V mutant(one of them had both M550V and L526M mutations).Conclusion The method of the present study was demonstrated to be an easy way to detect HBV lamivudine resistant mutants and can be applied to clinical monitoring of lamivudine resistance.