目的 筛选和鉴定幽门螺杆菌(Helicobacter pylori,Hp)中性粒细胞激活蛋白(neutrophil-activating protein,NAP)的有效抗原表位,为Hp疫苗的研制提供基础.方法 以抗NAP的单克隆抗体作为固相筛选分子,经3轮吸附-洗脱-扩增免疫,筛选噬菌体随机7肽库,随机挑选噬菌体克隆,经噬菌体酶联免疫吸附试验(ELISA)、交叉反应试验及竞争抑制试验鉴定阳性克隆,测定阳性克隆所携带DNA序列并进行计算机辅助分析.以制备的阳性噬菌体克隆短肽液免疫小鼠,免疫血清与NAP经Western blot分析,以验证NAP的模拟表位.结果 经3轮免疫筛选后挑选到45个阳性克隆,经ELISA鉴定有12个阳性克隆,测序结果显示5种表位,其中P17噬菌体展示肽FAHLATQ与NAP氨基酸序列(137~143)高度同源,位于NAP高抗原区域(118~140),免疫血清可识别NAP.结论 用噬菌体随机7肽库成功筛选到了NAP的模拟表位,为基于NAP的诊断和疫苗的研制提供了基础.%Objective To identify epitope of neutrophil-activating protein(NAP) of Helicobacter pylori(Hp).Methods Using the mouse monoclonal antibodies against NAP as selective molecular and immunoscrcening phage-display random 7-peptides library.The positive clones were sequenced and analyzed.Phage clones were chosen to immunize mice and to evaluate the potential of phagotopes as effective vaccines.Results One mimotope(FAHLATQ)showed a good match with the NAP at 140-143 AA(AHLA)and the serum of mice induced by the phage clone clearly recognized NAP.Conclusion This study suggests thatthe antigenic epitope could be mapped through screening the phage-display peptide library with monoclonalantibody and a mimotopo of NAP providing an ahernative approach for the diagnosis and development of avaccine for Hp.
展开▼