Construction of a replication-competent hepatitis B virus vector carrying secreted luciferase transgene and establishment of new hepatitis B virus replication and expression cell lines
Construction of a replication-competent hepatitis B virus vector carrying secreted luciferase transgene and establishment of new hepatitis B virus replication and expression cell lines
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携带分泌型荧光素酶转基因的具有复制能力的乙型肝炎病毒载体的构建及新型乙型肝炎病毒复制表达细胞系的建立
DOI：
10.3748/wjg.v25.i39.5961
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发表时间：
2019-10
期刊：
影响因子：
--
通讯作者：
孙殿兴
中科院分区：
文献类型：
--
作者：
阮杰;平采艳;孙烁;孙殿兴
背景此前，我们通过将P开放阅读框（ORF）从前C/C ORF中解偶联，精心设计转基因插入位点，成功构建了具有复制能力的乙型肝炎病毒（HBV）载体，以克服HBV基因组的紧凑组织并保持HBV复制能力。成功获得携带外源基因的具有复制能力的HBV载体，包括携带杀稻瘟菌素抗性基因（399 bp）的pCH-BsdR和携带人源化海肾绿色荧光蛋白基因（720 bp）的pCH-hrGFP。但前者的复制效率较高，但使用繁琐，而后者的复制效率较差且无法量化。因此，我们需要寻找一种方便且可量化的新报告基因以供进一步研究。目的建立细胞内乙型肝炎病毒复制和抗病毒药物筛选研究的有用工具。方法利用本实验室构建的具有复制能力的HBV病毒载体，结合分泌型荧光素酶报告基因，构建表达分泌型Nanoluc荧光素酶报告基因（SecNluc）的具有复制能力的HBV载体。该载体转染HepG2.TA2-7细胞，获得稳定分泌携带secNluc报告基因的HBV颗粒的细胞系。结果携带SecNluc报告基因pCH-sNLuc的具有复制能力的HBV载体可以产生所有主要病毒RNA和全套包膜蛋白，并实现高水平的分泌型荧光素酶表达。乙型肝炎病毒复制中间体可以从该载体产生。通过pTRE-sNLuc转染和潮霉素筛选，我们获得了分离的细胞克隆，命名为HBV-NLuc-35细胞，该细胞能够分泌secNLuc重组病毒，并且对现有的抗HBV药物敏感。使用分化的HepaRG细胞，证实重组HBV具有感染性。结论我们的研究表明，携带分泌型荧光素酶转基因的具有复制能力的HBV载体具有复制和表达能力，并且建立的HBV复制和表达细胞系可以稳定分泌携带secNluc报告基因的病毒颗粒。更重要的是，细胞系和分泌的重组病毒颗粒可用于追踪乙型肝炎病毒的复制或感染。
BACKGROUND Previously, we have successfully constructed replication-competent hepatitis B virus (HBV) vectors by uncoupling the P open reading frame (ORF) from the preC/C ORF to carefully design the transgene insertion site to overcome the compact organization of the HBV genome and maintain HBV replication competence. Consequently, the replication-competent HBV vectors carrying foreign genes, including pCH-BsdR, carrying blasticidin resistance gene (399 bp), and pCH-hrGFP, carrying humanized renilla green fluorescent protein gene (720 bp), were successfully obtained. However, the replication efficiency of the former is higher but it is tedious to use, while that of the latter is poor and cannot be quantified. Hence, we need to search for a new reporter gene that is convenient and quantifiable for further research. AIM To establish a helpful tool for intracellular HBV replication and anti-viral drugs screening studies. METHODS We utilized the replication-competent HBV viral vectors constructed by our laboratory, combined with the secreted luciferase reporter gene, to construct replication-competent HBV vectors expressing the reporter gene secretory Nanoluc Luciferase (SecNluc). HepG2.TA2-7 cells were transfected with this vector to obtain cell lines with stably secreted HBV particles carrying secNluc reporter gene. RESULTS The replication-competent HBV vector carrying the SecNluc reporter gene pCH-sNLuc could produce all major viral RNAs and a full set of envelope proteins and achieve high-level secreted luciferase expression. HBV replication intermediates could be produced from this vector. Via transfection with pTRE-sNLuc and selection by hygromycin, we obtained isolated cell clones, named HBV-NLuc-35 cells, which could secrete secNLuc recombinant viruses, and were sensitive to existing anti-HBV drugs. Using differentiated HepaRG cells, it was verified that recombinant HBV possessed infectivity. CONCLUSION Our research demonstrated that a replication-competent HBV vector carrying a secreted luciferase transgene possesses replication and expression ability, and the established HBV replication and expression cell lines could stably secrete viral particles carrying secNluc reporter gene. More importantly, the cell line and the secreted recombinant viral particles could be used to trace HBV replication or infection.
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影响因子：
3.7
作者：
Edmonds TG;Ding H;Yuan X;Wei Q;Smith KS;Conway JA;Wieczorek L;Brown B;Polonis V;West JT;Montefiori DC;Kappes JC;Ochsenbauer C
通讯作者：
Ochsenbauer C
通过重新设计的病毒聚合酶翻译，具有复制能力的传染性乙型肝炎病毒载体携带大量转基因。
影响因子：
3.7
作者：
Wang Z;Wu L;Cheng X;Liu S;Li B;Li H;Kang F;Wang J;Xia H;Ping C;Nassal M;Sun D
通讯作者：
Sun D
影响因子：
64.8
作者：
Stephen Hess
通讯作者：
Stephen Hess
DOI：
10.1073/pnas.84.4.1005
发表时间：
1987-02-01
影响因子：
11.1
作者：
SELLS, MA;CHEN, ML;ACS, G
通讯作者：
ACS, G
更正
DOI：
10.1136/bjsm.2006.033563cor1
发表时间：
1994
期刊：
The Lancet
影响因子：
--
作者：
J. Smeds;J. George;V. Vega;Liza Vergara;A. Ploner;Y. Pawitan;Per Hall;S. Klaar;E. Liu;J. Bergh
通讯作者：
J. Bergh