登革病毒包膜EDⅢ蛋白广谱中和抗体识别表位鉴定  

Identification of epitopes recognized by broad spectrum neutralizing antibodies against envelope protein domain Ⅲ of dengue virus

作　　者：易海粟[1] 温坤[1] 朱伟[1] 陈静[1] 车小燕[1] 富宁[1] 吴玉章[1] 

机构地区：[1]南方医科大学珠江医院临床免疫中心检验医学部,广州510282

出　　处：《免疫学杂志》2015年第7期612-617,共6页Immunological Journal

基　　金：国家自然科学基金(81201296)

摘　　要：目的分析登革病毒广谱中和抗体识别EDⅢ蛋白的表位特征。方法以对4个血清型登革病毒具交叉中和保护作用的单抗为钓饵筛选噬菌体展示肽库,用ELISA鉴定与单抗结合的噬菌体克隆,提取阳性噬菌体克隆的DNA测序,对测序结果进行生物信息学分析,并选择阳性噬菌体克隆制备抗血清鉴定噬菌体展示肽的抗原性。结果获得了与单抗2B11A35和2D73A7结合的噬菌体展示肽克隆,并证实其模拟EDⅢ蛋白的构象性表位,生物信息学分析提示单抗2B11A35和2D73A7识别EDⅢ蛋白EF loop附近区域;用2B11A35筛选噬菌体克隆免疫小鼠所获抗血清可与登革1、3及4型EDⅢ蛋白及2型病毒感染细胞结合。结论发现了1个EDⅢ蛋白上保守的保护性构象表位,为登革热疫苗的设计提供了有效组分。This study performed to identify and characterize the epitopes recognized by cross-neutralizing antibody against envelope protein domain m （ED Ⅲ ） of dengue virus （DENV）. Two strains of cross-neutralizing monoclonal antibody （mAb） against ED m of dengue virus were used to screen the epitopes from phage peptide library, and the selected phage clones were tested by sandwich enzyme-linked immunosorbent assay （ELISA）, and then analyzed by sequencing and bioinformatics. Furthermore, the antigenicity of the mimotope to conservative epitope within ED Ⅲ was identified by murine antisera immunized with positive phage clones. Phage clones binding to mAb 2BllA35 and 2D73A7 were obtained and proved as conformational epitopes. The bioinformatics analysis showed that mAb 2B11A35 and 2D73A7 could recognize the epitope that located in the EF loop on ED Ⅲ protein. The antisera of mice immunized with 3 phage clones binding to mAb 2B11A35 were capable of binding to dengue 1, 3 and 4 ED Ⅲ proteins and DENV 2 infected ceils. In conclusion, we identified a new conservative and protective epitope in ED Ⅲ protein recognized by two strains of cross-neutralizing mAbs, which could be used for dengue virus vaccine design.

关 键 词：登革病毒 中和抗体 噬菌体展示肽库 表位 包膜蛋白 

分 类 号：R373.1[医药卫生—病原生物学]