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  

Phenotypic characteristics of a novel mutation rtM204Q in the reverse-transcriptase domain of hepatitis B virus isolated from a chronic hepatitis B patient receiving lamivudine treatment

作　　者：王嫣[1,2] 刘妍[2] 许智慧[2] 思兰兰[2] 陈丽[2] 戴久增[2] 姚增涛[2] 徐东平[2] 

机构地区：[1]桂林医学院,广西桂林541004 [2]解放军302医院全军传染病研究所/肝衰竭诊疗与研究中心病毒性肝炎研究室

出　　处：《解放军医学杂志》2012年第6期548-551,共4页Medical Journal of Chinese People's Liberation Army

基　　金：国家“十二五”传染病重大专项子课题(2012ZX10004503)~~

摘　　要：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产物直接克隆到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rtA181T+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rtM204I对LAM的敏感性不及野生株的1/1000。rtM204Q和rtM204I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。Objective To analyze the phenotypic characteristics of a novel mutation rtM204Q in 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 nucleoside/nucleotide analogs（lamivudine： 0,0.01,0.1,1,10,100μmol/L;adefovir： 0,0.033,0.1,0.33,1,3.3μmol/L;entecavir： 0,0.001,0.01,0.1,1,10μmol/L;tenofovir： 0,0.01,0.1,1,10,100μmol/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%（P0.05） 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 compared to the wild type strain,while their susceptibility to adefovir,entecavir and tenofovir maintained unchanged.Conclusion The novel variation pattern rtM204Q in

关 键 词：肝炎病毒 乙型 抗药性 病毒 突变 

分 类 号：R512.62[医药卫生—内科学]