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

田青右,朱园飞,常豪,于琳,邓强

微生物与感染 ›› 2021, Vol. 16 ›› Issue (2) : 79-87.

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微生物与感染 ›› 2021, Vol. 16 ›› Issue (2) : 79-87. DOI: 10.3969/j.issn.1673-6184.2021.02.002
论著

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

  • 田青右,朱园飞,常豪,于琳,邓强
作者信息 +

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

  • TIAN Qingyou, ZHU Yuanfei, CHANG Hao, YU Lin, DENG Qiang
Author information +
文章历史 +

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EGFPN1-8/EGFPC9-11和sfGFPN1-10/sfGFPC11蛋白分裂系统。基于ΔHBc113载体,分别构建EGFPC9-11和sfGFPC11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EGFPC9-11和sfGFPC11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EGFPC9-11或sfGFPC11蛋白在共表达氮端蛋白EGFPN/sfGFPN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。

Abstract

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 of ΔHBc113 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 EGFPN1-8/EGFPC9-11 and sfGFPN1-10/sfGFPC11 split system, respectively. We further constructed EGFPC9-11 and sfGFPC11 rHBV replicons based on the ΔHBc113 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 EGFPN or sfGFPN, rHBV replicon-expressed EGFPC9-11 or sfGFPC11 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

Key words

Hepatitis B virus / Replicon;Intein / Green fluorescent protein / Split peptide

引用本文

导出引用
田青右,朱园飞,常豪,于琳,邓强. 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[J]. 微生物与感染. 2021, 16(2): 79-87 https://doi.org/10.3969/j.issn.1673-6184.2021.02.002
TIAN Qingyou, ZHU Yuanfei, CHANG Hao, YU Lin, DENG Qiang. Establishment of a recombinant hepatitis B virus replicon expressing a split green fluorescent protein[J]. Journal of Microbes and Infections. 2021, 16(2): 79-87 https://doi.org/10.3969/j.issn.1673-6184.2021.02.002
中图分类号: R373.2+1   

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