GⅡ.17型诺如病毒人源中和性单链抗体制备及生物特性鉴定  

Preparation and biological characterization of neutralizing scFv against Human GⅡ.17 Norovirus

作　　者：王璐 侯玉珍 曾志云 谢东杰 邱梦丝 张绪富(指导)[2] 戴迎春(指导)[1] WANG Lu;HOU Yu-Zhen;ZENG Zhi-Yun;XIE Dong-Jie;QIU Meng-Si;ZHANG Xu-Fu;DAI Ying-Chun(Department of Epidemiology,School of Public Health,Southern Medical University,Guangzhou 510515,China)

机构地区：[1]南方医科大学公共卫生学院流行病学系,广州510515 [2]南方医科大学中医药学院,广州510515

出　　处：《中国免疫学杂志》2021年第15期1860-1866,1871,共8页Chinese Journal of Immunology

基　　金：国家自然科学基金项目(31771007,81773975);广东省自然科学基金项目(2019A1515010951)。

摘　　要：目的:基于一起暴发疫情,构建人源噬菌体单链抗体库,筛选特异性GⅡ.17型诺如病毒(NoV)单链抗体(scFv),并进行表达纯化及鉴定。方法:采集患者粪便或肛拭子样本,RT-PCR检测肠道病毒核酸,PCR扩增NoV衣壳蛋白N/S区和P区序列,Mega 4.0构建系统进化树。采集并分离患者恢复期外周血PBMC,Trizol提取总RNA;RT-PCR扩增抗体重轻链可变区基因,重叠扩增PCR将两者拼接为scFv基因,将其克隆至噬菌粒载体pCANTAB-5E构建人源噬菌体单链抗体库。以GⅡ.17 P颗粒为固相抗原,对抗体库进行筛选及鉴定,将阳性克隆进行原核表达及纯化,ELISA鉴定其生物特性。结果:此次疫情中,83.3%(15/18)粪便或肛拭子样本呈GⅡ型NoV核酸阳性,经序列比对分析确定由GⅡ.17型感染导致;成功构建人源噬菌体单链抗体库,以GⅡ.17 P颗粒为抗原筛选得到11株序列不同且正确的阳性克隆;经原核表达获得9株与GⅡ.17 P颗粒具有良好亲和力的高纯度scFv,且与GⅡ.4和GI.3 P颗粒均有较好的特异性交叉反应,其中7株具有较强阻断GⅡ.17 P颗粒与HBGA受体结合的能力。结论:此次疫情为一次GⅡ.17型NoV引发的急性胃肠炎暴发,构建了人源噬菌体抗体库,经淘筛及表达纯化,最终获得9株GⅡ.17型NoV单链抗体,其中7株具有较强的中和能力。Objective:To construct a human phage antibody library based on an outbreak,to screen out specific single-chain variable fragments(scFv)against GⅡ.17 norovirus(NoV),and to express,purify and identify.Methods:Stool or anal swab samples of patients were collected,enterovirus nucleic acid was detected by RT-PCR,capsid protein N/S region and P region of NoV were amplified by PCR and phylogenetic tree was constructed by Mega 4.0.PBMC from peripheral blood of patients at convalescent phase were collected and isolated,and total RNA was extracted by Trizol.Variable heavy and light chains of antibodies were amplified by RT-PCR,and were assembled into scFv by splice overlap extension PCR,and was cloned to phagemid pCANTAB-5E to construct a phage antibody library.Antibody library was panned and identified taking P particle of GⅡ.17 NoV as an antigen,positive clones were subjected to sequence analysis,and were expressed and purified in a prokaryotic system,whose biological characterizations were identified by ELISA.Results:83.3%(15/18)of fecal or anal swab samples were positive for type GⅡNoV nucleic acid in this outbreak,and was determined from GⅡ.17 infection through BLAST analysis.A recombination phage antibody library against NoV was successfully constructed,and 11 clones with correct and different sequences were obtained after panning by GⅡ.17 P particle as antigen.9 strains of high-purity scFv with high affinity to GⅡ.17 P particle were obtained through prokaryotic expression,and all of them had a good cross-reaction with GⅡ.4 and GⅠ.3 P particles.Among them,7 strains showed rather strong ability to block binding of GⅡ.17 P particle to HBGA receptor.Conclusion:This outbreak of acute gastroenteritis was caused by GⅡ.17 NoV,and a human phage antibody library was constructed.After panning,expression and purification,9 strains of scFv aganist GⅡ.17 NoV were obtained,of which 7 strains showed rather strong neutralizing ability.

关 键 词：GⅡ.17型诺如病毒 噬菌体展示技术 人源单链抗体 

分 类 号：R392.11[医药卫生—免疫学]