接受拉米夫定治疗的慢性乙肝患者HBV反转录酶区新变异rtM204Q的表型特点分析

摘要

Objective To analyze the phenotypic characteristics of a novel mutation rtM204Qin the reverse-transcriptase (RT) domain of hepatitis B virus (HBV). Methods HBV DNA was isolated from a chronic hepatitis B patient receiving lamivudine treatment. The full-length RT region was amplified and the PCR product was cloned into the pGEM-Teasy vector. About 40 clones were randomly selected for DNA sequencing and drug-resistance-associated variation was analyzed. After restriction enzyme (Xho I /Sph I ) digestion and ligation procedure, the 1.1-ploid genome length HBV recombinant plasmids harboring wild type and three different mutants in RT region were constructed. Then the replication-competent constructs pTriEx-wRT and pTriEx-mRT were transiently transfected into the HepG2 cells by FuGene·HD transfection reagent. Four hours post-transfection, new media containing different concentrations of nudeoside/nucleotide analogs (lamivudine: 0, 0.01, 0.1, 1, 10, 100 μ mol/L; adefovir: 0, 0.033, 0.1, 0.33, 1, 3.3μmol/L; entecavir: 0, 0.001, 0.01, 0.1, 1, l0 μmol/L; tenofovir: 0, 0.01, 0.1, 1, 10, l00μ mol/L) were sequentially replaced every other day for 4 days. To analyze the phenotypic characteristics of the HBV mutant with different concentrations of the drug, the supernatant was collected and HBV DNA quantitation was done using real-time PCR. Results Sequence analysis of 37 clones showed that there were five mutants in RT region, 13 (35.1%) for wild type, 11 (29.7%) for rtM204Q, 2 (5.4%) for rtM204I, 10 (27.0%) for rtA181T, and 1 (2.7%) for rtA181T + rtM204Q. The relative replication capacity of the latter four mutants was 89.95%, 46.28%, 55.77% and 34.44% (P<0.05) of wild-type strain, respectively. Phenotypic resistance analysis of the former two mutants showed that susceptibility of rtM204Q and rtM204I strains to lamivudine was 1/75.68 and 1/1000 respectively so npared to the wild type strain, while their susceptibility to adefovir, entecavir and tenofovir maintained unchanged. Conclusion The novel variation pattern rtM204Q in HBV YQ DD motif could inhibit the replication capacity to some extent and present a less susceptibility to lamivudine, indicating that rtM204Q might be a novel lamivudine-resistant mutation.%目的 分析乙肝病毒(HBV)反转录酶(RT)区YMDD功能域新变异rtM204Q的表型特点.方法 从1例接受拉米夫定(LAM)治疗的慢性乙肝患者血清中提取HBV DNA,扩增HBV全长RT区基因,PCR产物直接克降到pGEM-Teasy载体中,随机挑选40个克隆进行DNA序列测定并分析耐药相关变异.将Xho I/Sph I双酶切后的RT片段与载体连接成含HBV 1.1倍基因组长度的重组载体.采用FuGENE·HD试剂盒,将构建的含野生株和变异株的重组载体转染至人肝癌细胞系HepG2细胞.转染4h后加入不同浓度的核苷(酸)类药物[LAM终浓度为0、0.01、0.1、1、10、100μmol/L;阿德福韦酯(ADV)终浓度为0、0.033、0.1、0.33、1、3.3μmol/L;恩替卡韦(ETV)终浓度为0、0.001、0.01、0.1、1、10μmol/L；替诺福韦酯(TDF)终浓度为0、0.01、0.1、1、10、100μmol/L],隔天换药,连续作用4d后收集细胞上清,采用实时荧光定量PCR技术检测不同药物浓度下HBV DNA的复制水平,以分析变异株的表型特点.结果 对从患者血清获得的37个克隆进行测序分析,结果显示13个(35.1％)为野生型,11个(29.7％)为rtM204Q突变型,2个(5.4％)为rtM204I突变型,10个(27.0％)为rtA181T突变型,1个(2.7％)为rtA18 4T+rtM204Q突变型.后4种变异株的复制力分别是野生株复制力的89.95％、46.28％、55.77％和34.44％(P＜0.05).前2种变异形式的表型耐药分析结果显示,rtM204Q对LAM的敏感性为野生株的1/75.68,而rtM2041对LAM的敏感性不及野生株的1/1000.rtM204Q和rtM2 041株对ADV、ETV和TDF均敏感.结论 HBV RT区新的YQDD基序变异在一定程度上抑制病毒复制力,对LAM的敏感性降低,提示这可能是一个新的LAM耐药相关变异.