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首页> 外文期刊>World Journal of Gastroenterology >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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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

机译：携带分泌荧光素酶转基因的复制型乙型肝炎病毒载体的构建及新乙型肝炎病毒复制的建立及表达细胞系

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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.?The Author(s) 2019. Published by Baishideng Publishing Group Inc. All rights reserved.
机译：背景：通过解偶联来自前C / C ORF在P开放阅读框（ORF）仔细以前，我们已经成功地构建了具有复制能力的乙型肝炎病毒（HBV）的载体设计转基因插入位点，以克服HBV基因组的紧凑组织并维持HBV复制的能力。因此，有复制能力的HBV载体携带外源基因，包括PCH-BsdR，杀稻瘟菌素携带抗性基因（399个碱基），和PCH-hrGFP的多克隆，携带人源化海肾绿色荧光蛋白基因（720个碱基），成功地获得。然而，前者的复制效率较高，但它是乏味的使用，而后者的差，无法量化。因此，我们需要寻找一个新的报告基因是方便和可量化的进一步research.AIM：建立细胞内HBV复制和抗病毒的药物筛选studies.METHODS一个有用的工具：我们使用的复制能力的HBV病毒载体由我们的实验室，与分泌的萤光素酶报告基因构建合并，以构建表达报告基因分泌Nanoluc荧光素酶（SecNluc）复制能力的HBV载体。 HepG2.TA2-7细胞用此载体中以获得与携带secNluc记者gene.RESULTS稳定分泌HBV颗粒的细胞系：携带SecNluc报道基因PCH-sNLuc有复制能力的HBV载体能产生所有主要的病毒RNA和全包膜蛋白的组，实现高水平的分泌萤光素酶表达。 HBV复制中间体可从该载体中来制造。通过与pTRE-sNLuc转染和选择潮霉素，我们获得的分离细胞克隆，命名为HBV-NLuc-35细胞，它可以分泌secNLuc重组病毒，并且是对现有的抗HBV药物敏感。使用分化的HepaRG细胞，它证实了重组HBV具有infectivity.CONCLUSION：我们的研究表明，携带荧光素酶分泌转基因的具有复制能力的HBV载体具有复制和表达的能力，以及所建立的HBV复制和表达的细胞系中能稳定分泌的病毒粒子携带secNluc报告基因。更重要的是，细胞系和分泌的重组病毒颗粒可以被用来跟踪HBV复制或infection.?The作者（一个或多个）2019年出版保留Baishideng出版集团所有权利。

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