乙型肝炎病毒rtA181V/T和rtN236T变异阿德福韦耐药株表达质粒的构建及其病毒学特征  

The construction of HBV strains with rtN236T and rtA181V/T mutations associated with resistance to adefovir and its viral characteristics

作　　者：王江华[1] 李俊强[1] 蒋栋[1] 费然[1] 丛旭[1] 杜绍财[1] 魏来[1] 

机构地区：[1]北京大学人民医院 北京大学肝病研究所,100044

出　　处：《中华检验医学杂志》2007年第9期1035-1039,共5页Chinese Journal of Laboratory Medicine

基　　金：国家自然科学基金(30571639);国家重点基础研究发展规划资助项目(2005CB522902)

摘　　要：目的构建乙型肝炎病毒阿德福韦耐药变异株(rtA181T/V 和 rtN236T)表达载体并对其体外复制能力和耐药性等病毒学特征进行体外研究。方法以含1.2倍拷贝 HBV DNA 全基因的质粒 PUC-HBV1.2WT为模板,PCR 定点诱变技术构建含阿德福韦耐药株(rtA181V/T和 rtN236T)的目的质粒,测序验证并利用变异株特异检测引物进行 PCR 检测;转染人肝癌细胞系 HepG2,ELISA 检测上清中分泌的 HBsAg 和 HBeAg 水平,荧光定量 PCR 检测上清中病毒 DNA 水平,Southern blotting 检测胞浆 HBV 复制中间体水平,比较野生株和变异株体外复制能力和对阿德福韦敏感性的差异。结果构建的 rtA181T、rtA181V 和 rtN236T 表达质粒可用于变异株特异检测引物检测相应变异的阳性对照品;转染 HepG2细胞后均可获得高水平的病毒抗原表达,胞浆和细胞培养上清中可以检测到 HBV 复制中间体和病毒颗粒的存在;三种变异均可单独导致对阿德福韦耐药,IC_(50)为野生株的2.8至4.7倍;体外复制能力较野生株降低,分别为野生株的94.2%、89.0%和77.7%。结论成功构建乙型肝炎病毒阿德福韦变异株表达质粒并应用于变异株特异引物扩增检测技术,体外实验证实 rtA181V/T 和rtN236T 单独变异均可导致 HBV 对阿德福韦耐药,且变异株的病毒复制能力较野生株有所下降。Objective To construct expression vector containing HBV rtN236T and rtA181V/T mutations and explore their replication capacity and susceptibility to adefovir after transient transfection into HepG2 cells. Methods The plasmid containing the entire HBV （ 1.2 copies） genome was used to construct adefovir-resistant expression plasmids via PCR-based site-directed mutagenesis in vitro. The constructed plasmids were validated by DNA sequencing and served as positive controls for mutant-specific PCR. After transient transfection into HepG2 cells, HBsAg and HBeAg in supematants were measured by ELISA, HBV DNA levels in supematants and Intracellular HBV replicative intermediates were measured by Florescence Real-time PCR and Southern blotting, respectively To compare the replication capacity and susceptibility to adefovir. Results Plasmids containing adefovir resistance mutations of rtA181T/V or rtN236T were constructed and expressed in HepG2 cells. These plasmids can be specifically positive control for mutantspecific PCR. In the transfection experiment, mutation with rtA181T/V or rtN236T could independently confer resistance to adefovir in vitro, IC50 of which were 2. 8 to 4. 7 folds more than wild type. Also, rtA181T/V or rtN236T mutants showed decreases of replication capacities, which were 94. 2% ,89.0% and 77. 7% of wild type HBV, respectively. Conclusion HBV with either rtA181T/V or rtN236T can confer resistance to adefovir resulting in decreased replication capacity.

关 键 词：肝炎病毒 乙型 阿德福韦 突变 

分 类 号：R512.6[医药卫生—内科学]