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  

作　　者：Jie Ruan Cai-Yan Ping Shuo Sun Xin Cheng Peng-Yu Han Yin-Ge Zhang Dian-Xing Sun 

机构地区：[1]The Liver Disease Center of Chinese People’s Liberation Army,the 980th Hospital of Chinese People’s Liberation Army Joint Logistics Support Force,Shijiazhuang 050082,Hebei Province,China [2]Department of Infection and Liver Disease,Shannxi University of Chinese Medicine Affiliated Hospital,Xianyang 712000,Shannxi Province,China

出　　处：《World Journal of Gastroenterology》2019年第39期5961-5972,共12页世界胃肠病学杂志（英文版）

基　　金：Supported by the National Natural Science Foundation of China,No.81672041;the National Major Science and Technology Special Project for Infectious Diseases of China,No.2012ZX10004503-012

摘　　要：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 pCHsNLuc 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 expressionBACKGROUND 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 sec Nluc reporter gene.RESULTS The replication-competent HBV vector carrying the SecNluc reporter gene p CHs NLuc 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 sec NLuc recombinant viruses, and were sensitive to existing anti-HBV drugs. Using differentiated Hepa RG cells, it was verified that recombinant HBV possessed infectivity.CONCLUSION Our research demonstrated that a replication-competent HBV vector carrying a secreted luciferase transgene posses

关 键 词：HEPATITIS B VIRUS Replication-competent HEPATITIS B VIRUS vector SECRETED LUCIFERASE gene HEPATITIS B VIRUS cell line 

分 类 号：R51[医药卫生—内科学]