乙型脑炎病毒疫苗株包膜蛋白K138E回复突变对其神经毒力的影响  

The effect on neurovirulence of a Japanese encephalitis virus vaccine strain with a reverse mutation of the envelope protein K138E

作　　者：袁磊 唐丽萍 黄荣[1] 冯亚岚 杨会强 杨健[1] 李玉华[3] YUAN Lei;TANG Li-ping;HUANG Rong;FENG Ya-lan;YANG Hui-qiang;YANG Jian;LI Yu-hua(School of Basic Medical Sciences,North Sichuan Medical College,Nanchong,Sichuan,China 637000;Department of Viral Vaccines,Chengdu Institute of Biological Products;Department of Arbovirus Vaccines,National Institutes for Food and Drug Control)

机构地区：[1]川北医学院基础医学院,四川南充637000 [2]成都生物制品研究所病毒疫苗研究室 [3]中国食品药品检定研究院虫媒病毒研究室

出　　处：《中国病原生物学杂志》2019年第7期790-795,共6页Journal of Pathogen Biology

基　　金：四川省教育厅重点项目(No.15ZA0205);南充市市校合作项目(No.NSMC20170406)

摘　　要：目的探索乙型脑炎疫苗株包膜蛋白(E蛋白)138位氨基酸向野毒株回复突变对其神经毒力与神经侵袭力的影响。 方法采用重叠延伸PCR技术扩增含有K138E突变位点的DNA片段,构建乙脑疫苗株E蛋白K138E位氨基酸突变的病毒感染性克隆,体外转录病毒RNA,电转染BHK21细胞拯救病毒,绘制病毒生长曲线,采用蚀斑试验与测序鉴定突变病毒,通过动物试验评价突变病毒与疫苗株对昆明小鼠的神经毒力和神经侵袭力。 结果酶切和测序表明K138E突变病毒感染性克隆构建成功,并通过电转染BHK21细胞包装得到K138E突变病毒。蚀斑试验显示突变病毒的蚀斑稍大于疫苗株,两株病毒的生长曲线与神经侵袭力无明显不同,但突变病毒神经毒力显著升高。 结论乙脑疫苗株E138位氨基酸回复突变后神经毒力增强,E138位氨基酸是调控疫苗株神经毒力的重要位点。Objective To investigate the effect of a reverse mutation at amino acid 138(K138E)of the envelope protein of a Japanese encephalitis vaccine strain on the virulence of that Japanese encephalitis vaccine strain. Methods A DNA fragment containing the K138Emutation site(prM/E(K138E)was obtained via overlapping extension PCR.The fragment was then inserted into the pACNR-JEV(F1+F3)plasmid to construct an infectious clone of the K138Erevertant virus.The viral RNA obtained via transcription in vitro was transfected into BHK21cells via electroporation to package the virus.The mutant virus K138Ewas identified using aplaque test and sequencing,and a viral growth curve was plotted.The neurovirulence and the neuroinvasiveness of the recovered mutant virus K138Eand the vaccine strain SA14- 14-2were compared in mice. Results Restriction enzyme digestion and sequencing indicated that an infectious clone of the K138Erevertant virus was successfully constructed.Sequencing indicated that the K138Erevertant virus was obtained via electroporation into BHK21cells.The plaque size of the revertant virus(3.2mm,average diameter)was slightly larger than that of the vaccine strain(2.7mm,average diameter).The growth curves of the two viruses were similar,and the titers of both viruses peaked 48hours post-infection.Animal experiments indicated that the neurovirulence of the K138Erevertant virus(LD50=2.62log10PFU,i.c.route)was significantly stronger than that of the vaccine strain (LD50≥5.74log10PFU,i.c.route),but the same was not true for neuroinvasiveness. Conclusion E138is a critical amino acid site that controls the neurovirulence of a Japanese encephalitis virus vaccine strain.

关 键 词：乙型脑炎 回复突变 神经毒力 感染性克隆 

分 类 号：R373.31[医药卫生—病原生物学]