蓝舌病病毒17型VP2蛋白单克隆抗体的制备及其抗原表位的鉴定  

Preparation of the monoclonal antibodies against VP2 protein of bluetongue virus serotype 17 and the VP2 B-cell epitope identification

作　　者：王凌凤[1] 杨涛[1] 孙恩成[1] 耿宏伟[1] 秦永丽[1] 赵晶[1] 蔡绪禹[1] 刘霓红[1] 李文京[2] 吴东来[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/农业部兽医公共卫生重点开放实验室,黑龙江哈尔滨150001 [2]中国动物疫病预防控制中心,北京100125

出　　处：《中国预防兽医学报》2011年第6期465-470,共6页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家863项目(2007AA02Z406);黑龙江省自然科学基因项目(ZJN-0602-01)

摘　　要：为制备蓝舌病病毒(BTV)血清17型VP2蛋白的单克隆抗体(MAb)及鉴定其抗原表位,本研究用原核表达系统部分重叠表达的两段VP2蛋白共同免疫BALB/c小鼠,采用细胞融合技术获得杂交瘤细胞,通过以重组VP2蛋为白包被抗原的间接ELISA筛选获得2株稳定分泌抗BTV17 VP2蛋白的MAbs杂交瘤细胞株,分别命名为3F4和4H10。Ig亚类鉴定2株MAbs均为IgG1/κ链。Western blot证明,2株MAbs均能识别重组VP2蛋白。间接免疫荧光试验表明:2株MAbs均与BTV17呈阳性反应,其中MAb 3F4与BTV1、BTV2、BTV3、BTV5、BTV8、BTV11、BTV13、BTV16、BTV23、茨城病病毒(IBAV)、牛轮状病毒(BRV)、牛呼肠孤病毒(RV)均呈阴性反应,但与BTV10和BTV24呈弱阳性反应。利用合成多肽对VP2抗原表位鉴定结果表明,MAb 3F4识别的抗原表位为540DPWNNR545,MAb 4H10识别的抗原表位为540DPWNNRA546。本研究结果为建立BTV17型特异性检测方法及VP2功能研究奠定了基础。In this study,monoclonal antibodies（MAbs） against bluetongue virus（BTV） serotype 17 were developed by fusion the myeloma cells with spleen cells from BALB/c mice immunized with purified 2 partial overlapping recombinant VP2 of BTV17.Two hybridoma cell lines stably secreting MAbs against VP2 of BTV17,designated 3F4 and 4H10,were identified by indirect ELISA using the recombinant VP2 as coating antigen.Both of MAbs could react with recombinant VP2 by western blot.Indirect immunofluorescence assay indicated that both of MAbs could react positively with BTV17,but MAb 3F4 was showed weakly positive with BTV10 and BTV24,whereas no cross reaction was found for BTV1,BTV2,BTV3,BTV5,BTV8,BTV11,BTV13,BTV16,BTV23,Ibaraki virus,bovine rotavirus and bovine reovirus.Further epitope mapping of BTV VP2 with a set of synthetic peptides showed that MAb 3F4 recognized the linear antigenic determinants of 540DPWNNR545 and MAb 4H10 reacted against 540DPWNNRA546 on the VP2 of BTV17,respectively.The preparation of MAbs and identification of VP2 epitopes might have potential applications in BTV serotype-specific detection method establishment,and structural and functional analysis of VP2 of BTV17.

关 键 词：蓝舌病病毒血清17型 VP2 原核表达 单克隆抗体 抗原表位 

分 类 号：S852.65[农业科学—基础兽医学]