摘要
目的建立诺如病毒原型株诺瓦克病毒(Norwalk virus,NV)P蛋白细菌细胞表面展示体系,并对其进行功能评价。方法构建pET28a-inaQn-TB-P(GI.1)重组质粒,并在大肠杆菌中进行诱导表达,建立升级版假NV。以猪胃粘膜提取物中已知NV受体组织血型抗原(human histo-blood group antigens,HBGAs)为对象,验证假病毒特异性识别、捕获HBGAs的能力;以生菜叶中类HBGAs为对象,验证升级版假NV捕获生菜叶中NV配体的特性,并对所捕获的配体进行初步分析。结果获得可高效捕获NV受体/配体的假病毒体系,该体系可特异性识别并捕获猪胃粘膜提取物中A型HBGA和生菜中类HBGAs。另外,所捕获生菜中类HBGAs可以分别被A、H和Lewis a型HBGA单克隆抗体识别,且以H型HBGA抗原位点暴露最多。结论本研究成功建立了NV的P蛋白细菌细胞表面展示体系,可以识别并捕获HBGAs及其类似物,为解析NV与配体互作机制提供技术平台。
Objective To establish the bacterial surface display system of P protein from the prototypical human norovirus,Norwalk virus(NV),and to evaluate its function.Methods The recombinant plasmid pET28a-inaQn-TB-P(GI.1)was constructed and was induced and expressed in Escherichia coli to establish an upgraded mimic NV.The known NV receptors,histo-blood group antigens(HBGAs)in porcine gastric mucin(PGM)were used as the targets to test the ability of the mimic NV in specifically recognizing and capturing HBGAs.The HBGAs-like substance in romaine lettuce was used as the targets to test the ability of the upgraded mimic NV in capturing ligands of NV in romaine lettuce.The captured ligands were also analyzed.Results A bacterial surface display system of mimic NV with high capture efficiency was obtained.The system can specifically recognize and efficiently capture type A HBGA in PGM and HBGAs-like substance in romaine lettuce.In addition,HBGAs-like substance in romaine lettuce can be recognized by monoclonal antibodies against type A,H,and Lewis a HBGA,respectively.The type H HBGA had the most exposed antigenic sites.Conclusions In this study,the bacterial cell surface display system of NV P protein has been established successfully,which can specifically recognize and capture HBGAs and their analogs.This provides a technical platform for analyzing the interaction mechanism between NV and its ligands.
作者
徐梦
吴文瑾
徐婷
王大鹏
Xu Meng;Wu Wenjin;Xu Ting;Wang Dapeng(Department of Food Science and Technology,School of Agriculture and Biology,Shanghai Jiao Tong University,Shanghai 200240,China)
出处
《国际病毒学杂志》
2020年第2期92-96,共5页
International Journal of Virology
基金
国家自然科学基金(No.31772078)
上海交通大学交叉基金(Agri-X 2017005)。
关键词
诺瓦克病毒
细胞表面展示体系
P蛋白
生菜
配体
Norwalk virus
Bacterial surface display system
P protein
Romaine lettuce
Ligand
