摘要
[目的]本试验旨在探究日本脑炎病毒(Japanese encephalitis virus, JEV)非结构蛋白NS1的生物学功能。[方法]将JEV NS1基因克隆到pFastbac1载体并将其转化至感受态DH10Bac得到重组杆粒,将重组杆粒转染sf9细胞产生重组杆状病毒,通过病毒感染High5细胞成功表达了JEV NS1。在人脑微血管内皮细胞(HBMEC)培养液添加外源NS1蛋白,通过光学显微镜观察细胞形态,利用共聚焦显微镜观察JEV NS1蛋白对HBMEC表面唾液酸修饰的影响,IFA检测细胞中组织蛋白酶L的表达;小鼠经尾静脉注射JEV NS1及其N207糖基化位点突变体蛋白NS1 N207Q,分别于24、48和72 h通过伊文思蓝(EB)染色检测2个蛋白对小鼠的血脑屏障(BBB)通透性的影响。[结果]JEV NS1蛋白通过下调人脑内皮细胞表面的唾液酸修饰和上调组织蛋白酶L表达损伤内皮糖萼;动物试验表明NS1蛋白能够破坏小鼠的血脑屏障;NS1 N207Q蛋白处理对细胞表面唾液酸修饰的影响不显著,体内处理对小鼠的血脑屏障无明显损伤。[结论]JEV NS1蛋白具有损伤人脑微血管内皮细胞的生物学功能,突变N207位点可导致其损伤功能减弱,可为JEV亚单位疫苗候选抗原的优化提供新思路。
[Objectives]The purpose of this study was to investigate the biological functions of nonstructural protein 1(NS1)of Japanese encephalitis virus(JEV).[Methods]JEV NS1 gene was cloned into pFastbac1 vector and transformed into DH10Bac competent E.coli to generate a recombinant bacmid;sf9 cells were transfected with recombinant bacmid DNA to generate a recombinant baculovirus.JEV NS1 was successfully expressed by virus infection of High5 cells.Exogenous NS1 protein was added into the supernatant of human endothelial cells(HBMEC),cell morphology was observed using confocal microscopy,the effect of JEV NS1 protein on the sialic acid modification of HBMEC surface was observed,and the expression of tissue protease L in the cells was detected by IFA.Mice was injected with JEV NS1 and its N207 glycosylation site mutant protein NS1 N207Q via the tail vein,and the effect of these two proteins on the blood-brain barrier(BBB)permeability in mice were detected by Eoans blue staining(EB)at 24,48,72 hours.[Results]It was found that JEV NS1 protein could increase the expression of cathepsin L and lead to endodermic calyx injury by down-regulating sialic acid on human endothelial cells.Animal experiment showed that NS1 protein could disrupt the blood-brain barrier in mice.NS1 N207Q protein treated cells had no significant effect on the surface sialic acid modification,and in vivo treatment had no significant damage to the blood-brain barrier of mice.[Conclusions]JEV NS1 protein had the biological function of brain endothelial injury,and its damage function was greatly reduced after mutation of N207 site,which provided a new idea for the optimization of JEV subunit vaccine candidate antigen.
作者
潘俊慧
刘亚林
梁真洁
杨兴淼
谢盛达
曹瑞兵
PAN Junhui;LIU Yalin;LIANG Zhenjie;YANG Xingmiao;XIE Shengda;CAO Ruibing(College of Veterianry Medicine,Nanjing Agricultural University,Nanjing 210095,China)
出处
《南京农业大学学报》
CAS
CSCD
北大核心
2023年第6期1107-1115,共9页
Journal of Nanjing Agricultural University
基金
国家重点研发计划项目(2016YFD0500402)
国家自然科学基金项目(31772756)。
