抗马动脉炎病毒N蛋白单克隆抗体的制备及B细胞抗原表位的鉴定  

Preparation of the monoclonal antibodies against N protein of equine arteritis virus and identification of B cell epitopes of N protein

作　　者：赵世华[1] 戚亭[1] 郭巍[1] 田志革[1,2] 朱超[1,2] 相文华[1] 

机构地区：[1]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室/大动物研究室,黑龙江哈尔滨150001 [2]东北农业大学生命科学学院,黑龙江哈尔滨150030

出　　处：《中国兽医科学》2013年第10期1035-1039,共5页Chinese Veterinary Science

基　　金：公益性行业(农业)科研专项经费项目(201003075);中央级公益性科研院所基本科研业务费专项(0302012011)

摘　　要：为鉴定马动脉炎病毒(EAV)N蛋白的抗原表位,利用原核表达系统表达N蛋白,用其免疫6周龄BALB/c小鼠,将小鼠脾细胞与骨髓瘤SP2/0细胞融合获得杂交瘤细胞,通过以重组N蛋白为包被抗原的间接ELISA方法筛选出2株能稳定分泌抗N蛋白的单克隆抗体(MAb),分别将其命名为2B3和2B9,其重链属于IgG1,轻链属于κ链。Western-blot证明,2株MAbs均能识别重组N蛋白。间接免疫荧光(IFA)试验证明,2株MAbs均与EAV呈阳性反应。用肽扫描法将N基因截短为具有相互部分重叠的11段,表达带HIS标签的重组蛋白,与MAb 2B3和MAb 2B9进行Western-blot和ELISA反应后,针对表位序列11 ASFRNGRRRQPTSYND26,进一步通过合成短肽对2株MAbs进行精确定位;结果表明,2B3识别的抗原表位为18 RRQPTSYND26,2B9针对的抗原表位为18 RRQPTSYND26。本研究结果为建立EVA的血清学检测方法及研究N蛋白的结构和功能奠定了基础。To identify the epitopes of N protein,BALB/c mice of 6 week-old were immunized with N protein by expressed by the prokaryotic expression system and the spleen cells from the immunized mice were fused with mouse myelorna cells SP2/0. Two hybridoma cell lines 2B3 and 2B9,stably secreting MAbs against N protein,could react with EAV by Western-blot and by indirect immunofluorescence assay（IFA）. Epitopes of N protein were identified by pepscan technique. These two MAbs reacted with epitope ^18RRQPTSYND^26. These provide a foundation for the development of sero-diagnostic reagents and functional studies.

关 键 词：马动脉炎病毒 单克隆抗体 肽扫描 表位 

分 类 号：S852.659.6[农业科学—基础兽医学]