绿色荧光蛋白分裂肽基因重组乙型肝炎病毒复制子模型的建立  

Establishment of a recombinant hepatitis B virus replicon expressing a split green fluorescent protein

作　　者：田青右 朱园飞 常豪 于琳 邓强 TIAN Qingyou;ZHU Yuanfei;CHANG Hao;YU Lin;DENG Qiang(Department of Medical Microbiology and Parasitology,Key Laboratory of Medical Molecular Virology(MOE/NHC/CAMS),School of Basic Medical Sciences,Shanghai Medical College,Fudan University,Shanghai 200032,China)

机构地区：[1]复旦大学上海医学院基础医学院病原生物学系,教育部/卫健委/医科院医学分子病毒学重点实验室,上海200032

出　　处：《微生物与感染》2021年第2期79-87,共9页Journal of Microbes and Infections

摘　　要：为构建一种重组乙型肝炎病毒(hepatitis B virus,HBV)复制子模型,使其能够在病毒感染的细胞中表达可视化报告基因蛋白,本研究删除HBV基因组核心蛋白(HBV core,HBc)编码区部分序列,构建HBV1.1-ΔHBc113复制子载体。利用内含肽(intein)介导蛋白拼接的特性,选取加强绿色荧光蛋白(enhanced green fluorescent protein,EGFP)和超级折叠绿色荧光蛋白(super folder green fluorescent protein,sfGFP)作为外显肽,建立EGFP^(N1-8)/EGFP^(C9-11)和sfGFP^(N1-10)/sfGFP^(C11)蛋白分裂系统。基于ΔHBc113载体,分别构建EGFP^(C9-11)和sfGFP^(C11)重组HBV复制子;应用DNA印迹法(Southern blotting)验证两种rHBV载体的细胞内复制能力。结果显示,构建的HBV1.1-ΔHBc113复制子载体能够在HBc反式互补条件下在转染细胞中形成病毒复制。构建的EGFP^(C9-11)和sfGFP^(C11)重组HBV复制子能够支持HBc互补条件下的细胞内病毒复制,并产生子代病毒颗粒;HBV重组复制子表达的EGFP^(C9-11)或sfGFP^(C11)蛋白在共表达氮端蛋白EGFP^(N)/sfGFP^(N)细胞中,通过intein介导的蛋白质高效拼接,能够形成完整的、有功能的GFP。结果表明,构建的荧光蛋白重组HBV复制子系统能够为HBV高通量药物筛选和HBV易感性研究提供实验工具,具有重要的病毒学意义和应用前景。In the present study,we developed a recombinant hepatitis B virus(rHBV)replicon in order to visualize virus-infected living cells via reporter gene expression.We first constructed an HBV replicon vector HBV1.1-ΔHBc113,with partial deletion of the sequence encoding HBV core(HBc).The vector demonstrated a competent viral DNA replication in transfected cells,only if HBc was provided in trans.However,the replication efficiency ofΔHBc113 vector was greatly impaired by insertion of a large foreign DNA fragment.Taking advantage of the property of intein-mediated protein splicing,we chose enhanced-green fluorescent protein(EGFP)and super folder GFP(sfGFP)as exteins,and developed EGFP^(N1-8)/EGFP^(C9-11) and sfGFP^(N1-10)/sfGFP^(C11) split system,respectively.We further constructed EGFP^(C9-11) and sfGFP^(C11) rHBV replicons based on theΔHBc113 vector,the latter of which was proved to be competent for HBc-rescued,functional intracellular replication,and produced progeny virions in the culture medium.In cells co-expressing EGFP^(N) or sfGFP^(N),rHBV replicon-expressed EGFP^(C9-11) or sfGFP^(C11) was capable of working with their counterparts,respectively,forming intact and functional GFP through intein-mediated protein splicing.We thus successfully developed a fluorescent rHBV replicon system,which can be not only used for HBV infection mechanism study,but also have an extensive application prospect for high-throughput antiviral screening.

关 键 词：乙型肝炎病毒 复制子 内含肽 绿色荧光蛋白 分裂肽 

分 类 号：R373.21[医药卫生—病原生物学]