乙型肝炎病毒阿德福韦酯耐药株(RT A181T)感染性克隆的构建及在Huh7细胞中的表达  

Construction of infectious clone of HBV adefovir-resistant strain RT A181T and its expression in Huh7 cells

作　　者：赵建华[1] 陆仁飞[1] 

机构地区：[1]江苏省南通市第三人民医院检验科,226006

出　　处：《国际检验医学杂志》2016年第23期3266-3268,共3页International Journal of Laboratory Medicine

基　　金：江苏省南通市卫生和计划生育委员会资助项目(WQ2014038;W201217);江苏省南通市科技局资助项目(HS2014062)

摘　　要：目的利用重叠延伸聚合酶链反应(SOE-PCR)快速构建乙型肝炎病毒(HBV)阿德福韦酯耐药株(RT A181T)感染性克隆,观察重组质粒在Huh7细胞中的表达,建立HBV阿德福韦酯耐药株(RT A181T)的体外研究细胞模型。方法设计保守引物,从慢性乙型肝炎阿德福韦酯耐药患者血清中扩增全长HBV基因组,利用SOE-PCR技术构建1.3倍基因组长度的感染性克隆质粒pHBV1.3,转染肝癌细胞系Huh7,采Western Blot、Real-time PCR检测感染性克隆复制以及表达情况,同时用抗病毒药物拉米夫定以及阿德福韦酯验证对感染性克隆复制及表达的抑制情况。结果成功构建了HBV阿德福韦酯耐药株感染性克隆质粒pHBV1.3(RT A181T),该质粒在Huh7细胞系中能有效复制、转录和表达。拉米夫定能有效抑制该感染性克隆的复制和表达,阿德福韦酯不能抑制该感染性克隆的复制和表达。结论构建的HBV阿德福韦酯耐药株感染性克隆质粒pHBV1.3(RT A181T)在体外能高效复制及表达蛋白,其转染细胞可用于HBV复制机制及抗病毒研究。Objective To rapidly construct hepatitis B virus（HBV）adefovir（ADV）-resistant strain（RT A181T）infectious clone by using SOE-PCR,to observe the expression of recombinant plasmids in Huh7 cells and to establish the in vitro cell model of HBV ADV-resistant strain（RT A181T）.Methods The conservative primers were designed,the full-length HBV genome was amplified from serum in the patients with ADV-resistant chronic hepatitis B.Then,the 1.3-fold genome-length infectious clone pHBV1.3was constructed by using the SOE-PCR technique,which was transfected into human hepatoma cells Huh7 cell line.ELISA,Western Blot and Real-time PCR were adopted to detect infectious cloning replication and expression,meanwhile the antiviral drugs lamivudine（LAM）and ADV were used to verify the infectious clone copy and inhibition of expression.Results The HBV ADV-resistant infectious cloning plasmids pHBV1.3was successfully constructed.This plasmid could be effectively replicated,transcripted and expressed in Huh7 cell lines.LAM could effectively inhibit the replication and expression of the infectious clone,while ADV could not inhibit the replication and expression of the infectious clone.Conclusion The constructed infectious clone plasmids pHBV1.3（RT A181T）of HBV ADV-resistant strain can efficiently replicate and express protein in vitro,its transfected cells can be used in the study of HBV replication mechanism and antiviral study.

关 键 词：乙型肝炎病毒 阿德福韦酯 耐药 感染性克隆 

分 类 号：R512.62[医药卫生—内科学] R440[医药卫生—临床医学]