蓝舌病病毒3型VP5蛋白单克隆抗体的制备及其抗原表位鉴定  

Preparation of monoclonal antibody against VP5 of serotype3 bluetongue virus and identification of the antigenic epitope

作　　者：孙晶[1] 孙恩成[1] 刘二战[1] 孙亮[1] 徐青元[1] 杨涛[1] 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

出　　处：《中国预防兽医学报》2014年第5期387-390,共4页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家自然科学基金(31302065);中国博士后科学基金(2013M540172);公益性行业(农业)科研专项资助(201203056);新型重大动物疫苗与诊断试剂创制及生产工艺创新(2011AA10A213);兽医生物技术国家重点实验室基金(SKLVBP201316)

摘　　要：为制备血清3型蓝舌病病毒(BTV)VP5蛋白的单克隆抗体(MAb)及鉴定其抗原表位,本研究利用pMAL-C4x原核表达系统表达、纯化的重组VP5蛋白免疫BALB/c小鼠,取其脾淋巴细胞与SP2/0细胞进行融合,并以Bac-to-Bac昆虫杆状病毒表达系统表达、纯化的重组VP5蛋白为包被抗原,通过间接ELISA筛选出一株稳定分泌抗BTV3 VP5蛋白的MAb杂交瘤细胞株(3F2)。间接免疫荧光结果表明:MAb 3F2与BTV3、BTV5、BTV9、BTV16呈阳性反应;与BTV6、BTV7、BTV13、BTV21呈弱阳性反应;与其它BTV血清型均呈阴性反应。利用原核表达的麦芽糖结合蛋白(MBP)融合短肽对MAb 3F2识别的抗原表位进行鉴定,结果表明:该MAb识别的抗原表位为89PGERGIQMKIKEIEEE104。氨基酸序列比对结果显示该表位在不同地域来源的BTV3株间比较保守。该MAb的制备和鉴定为进一步研究VP5蛋白的结构和功能奠定了基础。For the preparation of monoclonal antibody （MAb） against protein VP5 of bluetongue virus （BTV） serotype 3 and identification of antigen epitope, SP2/0 myeloma cells were fused with spleen cells from BALB/c mice immunized with purified recombinant VP5 of BTV3 expressed in E.coli. One hybridoma cell lines stably secreting MAb against VP5 of BTV3 was identified by indirect ELISA coated with the purified recombinant VP5 expressed by baculovirus expression system. Indirect immunofluorescence assay indicated that the MAb had strong positive reaction with BTV3, BTV5, BTV9, BTV16, and weak reaction with BTV6, BTV7, BTV13 and BTV21, but no cross reaction with other BTV serotypes. Further antigen epitope identification for the MAb with a set of prokaryotic expressed maltose-binding protein （MBP）-fused overlapped short peptides showed that the MAb recognized the linear antigenic determinant of 89PGERGIQMKIKEIEEE104 on the VP5 of BTV3. These results demonstrated that preparation of the MAb would facilitate the further study on structure and founction of BTV3 VP5.

关 键 词：蓝舌病病毒3型 VP5蛋白 单克隆抗体 抗原表位 

分 类 号：S852.65[农业科学—基础兽医学]