用于抗西尼罗病毒药物筛选的假病毒报告系统的建立

Construction of a pseudovirus reporting system for anti-West Nile virus drug screening

目的为避免西尼罗病毒(WNV)研究中活病毒所带来的安全隐患,建立WNV的假病毒报告系统,用于抗WNV药物的筛选。方法构建含有海蜃荧光素酶报告基因的WNV复制子重组质粒prWNV-Rluc及表达WNV结构蛋白C、M和E的重组质粒pcDNA3.1-CME,并共转染293T细胞,72 h后获取含有假病毒的培养上清。结果用上清感染BHK21细胞后,BHK21细胞产生荧光,且荧光强度与感染细胞的假病毒量呈量效关系。Western印迹法实验检测到感染的BHK21细胞表达WNV NS1蛋白,证实上清中为WN假病毒。中和实验显示WNV中和性抗体能有效阻断WN假病毒对BHK21细胞的感染。结论建立了WNV的假病毒报告系统,规避了生物安全3级实验室(BSL-3)操作,可用于抗WNV药物的筛选。

[Abstact] ObjectiveTo establish a pseudovirus reporting system was established for anti-WNV drug screening in order to bypass security risk caused by live viruses in West Nile virus(WNV)research.Methods293 T Cells were co-transfected with WNV replicon recombinant plasmid pr WNV-Rluc containing Renilla luciferase reporter gene and recombinant plasmid pc DNA3.1-CME expressing WNV structural proteins C,M and E;and the cell culture supernatant containing pseudovirus was obtained at 72 h post-cotransfection.ResultsAfter pseudovirus infection with the supernatant,BHK21 cells produced fluorescence with the intensity that increased dose-dependently with the supernatant containing pseudovirus. The WNV NS1 Protein expressed in the infected BHK21 cells was identified by Western blot,confirming the presence of WN pseudovirus in the supernatant. Neutralization assay showed that the WNV neutralizing antibody could effectively block the infection of BHK21 cells by the WN pseudovirus in the supernatant.ConclusionThe West Nile pseudovirus reporting system established in this study might be used without the biosafety level 3 laboratory(BSL-3) and could be applied to the screening of anti-WNV drugs.