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目的:探索乙型脑炎病毒(JEV)疫苗株SA14-14-2包膜蛋白279位氨基酸(E279)MK回复突变(M279K)对其毒力的影响。方法:用重叠延伸PCR技术和基因克隆技术构建含有SA14-14-2包膜蛋白M279K突变的全长cDNA质粒pACNR-JEV(M279K),并以其为模板体外转录RNA,将RNA电转染导入BHK21细胞得到恢复病毒rJEV(M279K),并比较恢复病毒和疫苗病毒在蚀斑大小以及对小鼠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,除人为引入的突变外(碱基1813处T→A),无任何碱基突变,并发现rJEV(M279K)形成比疫苗株更小的蚀斑,但毒力与疫苗株无显著差异。结论:E279回复突变影响乙脑疫苗SA14-14-2病毒蚀斑大小,但并未明显增强疫苗株对小鼠的神经毒力。
Objective: To explore the effect of M279K on the virulence of the eukaryotic expression vector of 279 amino acid (E279) of SA14-14-2 envelope protein of Japanese encephalitis virus (JEV) vaccine strain. METHODS: The full-length cDNA plasmid pACNR-JEV (M279K) containing the M279K mutation of SA14-14-2 envelope protein was constructed by overlap extension PCR and gene cloning techniques. Transcribed RNA was used as a template to transcribe RNA into RNA The recovered virus rJEV (M279K) was obtained from BHK21 cells and the differences between the recovered virus and the vaccine virus in terms of plaque size and neurotoxicity in mice were compared. Results: The restriction enzyme digestion and sequencing showed that the plasmid template was successfully constructed. In addition to human introduced mutations (T → A at base 1813), no base mutation was found and rJEV (M279K) was found to form smaller plaques than the vaccine strain. However, there was no significant difference between virulence and vaccine strains. Conclusion: E279 back-mutation affects the size of SA14-14-2 virus plaque on JE vaccine, but does not significantly enhance the virulence of vaccine strain against mice.