乙型脑炎疫苗株SA14-14-2包膜蛋白279位氨基酸回复突变对其毒力的影响

M279K reversion in envelope protein: the effects on virulence of Japanese encephalitis vaccine strain SA14-14-2

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 investigate the effects on virulence of M279K reversion in envelope protein of Japanese encephalitis virus （JEV） vaccine SA14-14-2. Methods： The full-length cDNA harbouring M279K mutation of JEV SA14-14-2 was cloned into the low- copy plasmid pACNR with overlapping PCR and gene recombination technologies, the resulting plasmids were then identified with re- striction enzyme digestion and sequence analysis. RNA was transcribed from the plasmid template in vitro and electroporated into BHK21 cells, and the culture supernatant was collected and recovery virus rJEV （M279K） was detected with plaque assay and se- quence analysis. The form and size of the plaques and virulence of the 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 （M279K） containing M279K reversion was constructed successfully and sequence analysis showed that there was no mutant detected except an engineered mutation at nucleotide 1813 （T-,A） in the envelope protein of rJEV （M279K）. The plaque size of rJEV （M279K） in BHK21was smaller than that of vaccine strain, but the neurovirulence phenotype in Kun-miug mouse has no difference in rJEV （M279K） and vac- cine virus. Conclusion： M279K reversion affects the size of plaque of SA14-14-2 but not virulence.