乙型脑炎减毒活疫苗SAl4—14—2感染性克隆的构建、鉴定及稳定性分析

The construction, characterization and stability analysis of infectious clone of live attenuated Japa- nese encephalitis vaccine strain SA14-14-2 virus

目的构建稳定的乙型脑炎病毒（JEV）疫苗SAl4-14-2感染性克隆。方法用基因重组技术把SAl4-14—2全长cDNA克隆到质粒pACNR中，并以其为模板转录RNA，将RNA电转导入BHK21细胞包装病毒，用蚀斑实验和间接免疫荧光法检测病毒，对病毒进行传代实验及序列分析，并比较恢复病毒和疫苗株在蚀斑大小、神经毒力等方面的差别。结果酶切及测序表明质粒模板成功构建，用免疫荧光技术检测到恢复病毒蛋白表达，恢复病毒感染BHK21后可致细胞病变效应（CPE），传代实验表明病毒可以稳定感染BHK21细胞，测序表明：除人为引入的突变外（碱基473A—c）无任何碱基突变，并发现恢复病毒在蚀斑形成、小鼠的神经毒力等特性方面同疫苗株相同。结论成功构建了稳定的JEV感染性克隆，建立了用于JEV研究的反向遗传学方法，为研究JEV的致病机理、疫苗的减毒机制以及嵌合病毒疫苗的研发奠定基础。

Objective To construct a stable infectious clone of live attenuated Japanese encephali- tis virus （JEV） vaccine strain SA14-14-2. Methods Full-length cDNA of JEV was cloned into the low- copy plasmid pACNR with gene recombination technology and the resulting plasmids were identified with re- striction enzyme digestion and sequence analysis. RNA was transcribed from the plasmid template in vitro and electrotransfected into BHK21 cells, the culture supernatant was collected and recovery viruses were de- tected with plaque assay and the protein of recovery viruses was detected with immunofluorescence technolo- gy. The recovery viruses passaged in BHK21 cells and the sequence of recovery viruses was analyzed. The form and size of the plaque and neurovirulence of recovery viruses were compared with that of the parental strain SA14-14-2. Results Restriction enzyme digestion and sequence analysis showed that the plasmid pACNR-JEV containing the full-length cDNA of JEV SA14-14-2 was constructed successfully and the cDNA sequence is exactly loyal to the parental virus sequence. The recovery viruses induced cytopathogenic effect （CPE） in BHK21 cells and the envelope protein and non-structural protein 1 of recovery viruses was detec- ted in the infected LLC-MK2 cells with immunofluorescence assay. Recovery viruses and parent viruses were identical with respect to the plaque form, size in BHK21 cells, the neoruvirulence phenotype in Kun-ming mouse and nucleotide sequence except an artificial engineered mutation at nucleotide 473 （A---,C） in core gene. Conclusion The plasmid containing the full length cDNA of JEV was constructed, and the stable re- covery viruses were rescued successfully. The reverse genetics technology platform for JEV study was built, it will be significant for the studies of the pathogenesis of JEV, the genetics mechanism of virulence attenua- tion of vaccine strain SA14-14-2 and the developement of new chimeric vaccine of flavivirus.