重组水疱性口炎病毒载体病毒包装体系的建立及优化

摘要

Objective:To establish a stable and efficient recombinant vesicular stomatitis virus(VSV) packaging system for vaccine preparation,the recombinant VSV packaging system was investigated and optimized.Methods:Using prVSV△G-GFP as a scaffold vector,we explored the effects of different amount of scaffold and helper plasmids,different packaging cell lines,reaction time of recombinant poxvirus vTF7-3 and time of transfection on recombinant VSV packaging.Light microscope and fluorescence microscopy were used to determine the packaging effect of the recombinant VSV.The titer of the recombinant VSV was determined by TCID50 method.Results:The packaging efficiency of VSV decreased along with the decrease of the total amount of helper plasmids and packaging plasmids.When the amount of helper plasmids was constant,the packaging efficiency decreased along with the reduction of the scaffold plasmid.The packaging efficiency in 293T cells was more efficient than that of BHK21-WI2 cells.Proper reaction time and transfection time of the recombinant poxvirus vTF7-3 were necessary to increase the packaging efficiency of the VSV.The optimized rVSV△G-GFP packaging conditions were as follows:293T cells were infected by vTF7-3 at a multiplicity of infection(MOI) of 5～15 for 1 h or 5 MOI for 1～4 h,3.67～22 μg of total plasmids were used to transfect cells for 6～14 h.Finally,the packaged rVSV△G-ZE virus was successfully and efficiently established.Conclusion:A stable and efficient VSV packaging system was established,which lay the foundation for preparation of recombinant VSV vector vaccine.%目的:建立并优化重组水疱性口炎病毒(VSV)载体病毒包装体系,以获得稳定高效的重组VSV载体疫苗包装系统.方法:以prVSV△G-GFP为骨架载体,pBS-N、pBS-P、pBS-G、pBS-L为辅助载体,探索不同的骨架质粒及辅助质粒用量、包装细胞系、重组痘病毒vTF7-3作用时间、转染试剂作用时间等因素对重组VSV载体病毒包装的影响,用光学显微镜及荧光显微镜观测重组VSV载体病毒的包装效果,应用TCID50法测定重组VSV的滴度.结果:重组VSV的包装效率随着辅助质粒或包装质粒总量的减少而降低,293T细胞较BHK21-WI2细胞包装效率更高,适当延长重组痘病毒vTF7-3作用时间或转染试剂作用时间能提高VSV包装效率.优化的病毒包装条件为:选用293T细胞,vTF7-3以5～15 MOI感染细胞1h或以5 MOI感染细胞1～4 h,总质粒用量为3.67～22 μg,转染细胞6～14 h可获得高效包装的rVSV△G-GFP.采用优化包装条件高效包装了rVSV△G-ZE病毒.结论:获得了稳定高效的重组VSV包装体系,为重组VSV载体的疫苗研究奠定了基础.