带荧光素酶基因的乙型脑炎病毒SA14-14-2感染性克隆的构建及鉴定

目的：构建带有荧光素酶基因的乙型脑炎（简称乙脑）病毒感染性克隆，并通过体外拯救获得恢复病毒，用于乙脑病毒致病机理研究、病毒感染在小鼠体内的动态分布以及乙脑疫苗免疫效果评价的高通量方法建立。方法：应用反向遗传学、融合PCR以及无缝拼接等体外重组技术，将荧光素酶报告基因（F-LUC）插入到乙脑病毒SA14-14-2全长感染性克隆的5''非编码区与C蛋白编码区之间，线性化后体外转录为RNA并转染BHK21细胞，在细胞感染和动物试验分别检测恢复病毒的拯救和F-LUC的表达情况。结果：成功构建了带有F-LUC报告基因的SA14-14-2乙脑病毒全长感染性克隆，并拯救获得了重组病毒。基因序列分析及细胞与动物水平均证明拯救的病毒可较高水平表达荧光素酶基因。结论：本研究构建出带有荧光素酶报告基因F-LUC的SA14-14-2感染性克隆，并拯救获得带有荧光素酶的重组乙脑病毒，为乙脑血清中和抗体的高通量筛选、病毒感染在小鼠体内的动态分布和致病机理研究奠定了基础。

Construction of JEV SA14-14-2 Infectious Clone with Luciferase Gene

Objective:To construct Japanese encephalitis virus (JEV) SA14-14-2 infectious clone and rescue the JEV with luciferase gene, which is used for establishing a high-throughput method for studying pathogenesis of JEV, the dynamic distribution of JEV SA14-14-2 virus in mice, and the immune effectiveness of JE vaccine. Methods:Using reverse genetics and virus infectious cloning technology, the luciferase reporter gene F-LUC was inserted into the genome of JEV SA14-14-2 infectious clone to construct SA14-14-2 full-length infectious clones containing F-LUC, which was linearized and transcribed into RNA in vitro and transfected into BHK21 cells. Results:The full-length infectious clone of SA14-14-2 with F-LUC reporter gene was successfully constructed and recombinant virus was rescued. Gene sequence analysis and studies at cell and animal levels demonstrated that the rescued virus was a JEV with luciferase gene. Conclusion:JEV SA14-14-2 infectious clone with luciferase reporter gene F-LUC was constructed in this study, and the recombinant JEV with luciferase was rescued, which laid the foundation for the high-throughput screening of neutralizing antibody against JE serum, the dynamic distribution in mice and the pathogenesis of JEV.