蓝舌病病毒8型VP2蛋白单克隆抗体的制备及其抗原表位鉴定  

Preparation of the monoclonal antibodies against VP2 protein of bluetongue virus serotype 8 and the VP2 B-cell epitope identification

作　　者：席娜[1,2] 赵国辉[2] 孙恩成[2] 秦永丽[2] 孙亮[2] 魏天 徐青元[2] 杨涛[2] 孙晶[2] 刘二战[1,2] 钱爱东[1] 吴东来[2] 

机构地区：[1]吉林农业大学动物科学技术学院,吉林长春130000 [2]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4期318-322,共5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：为制备蓝舌病病毒03TV）8型VP2蛋白的单克隆抗体（MAb）TL鉴定其抗原表位，本研究采用Bac-to-Bac昆虫杆状病毒表达系统表达并纯化的重组VP2蛋白免疫BALB／c小鼠，三免后取小鼠脾淋巴细胞与SP2／0细胞进行融合，并以纯化的重组VP2蛋白为包被抗原，通过间接ELISA筛选出两株能够稳定分泌抗BTV8VP2蛋白的MAbs杂交瘤细胞株（2G4和387）。间接免疫荧光结果表明：2G4和387与BTV8均呈阳性反应，与BTV1～7、BTV9-24、茨城病毒、中山病毒以及赤羽病毒均呈阴性反应。Westernblot结果显示，2G4和387均能够识别BTV8及BTV8重组VP2蛋白。Ig亚类鉴定2G4和387均为IgG1／κ链。利用原核表达的96条覆盖VP2全长的麦芽糖结合蛋白（MBP）融合短肽对MAbs的抗原表位进行鉴定，结果表明：MAb2G4识别的抗原表位为捌LCRLLSTIGRKMCNTE^296。本研究结果为BTV8型特异性检测方法的建立及VP2蛋白结构和功能的研究奠定了基础。For the preparation of monoclonal antibodies （MAbs） against protein VP2 of bluetongue virus （BTV） serotype 8 and identification of the antigen epitope, SP2/0 myeloma cells were fused with spleen cells from BALB/c mice immunized with purified recombinant VP2 of BTV8 prepared by Bac-to-Bac insect baculovirus expression system. Two hybridoma cell lines stably secreting MAbs against VP2 of BTV8, named 2G4 and 3B7, were identified by indirect ELISA coated with the purifiedrecombinant VP2. Indirect immunofluorescence assay indicated that the two MAbs reacted positively with BTV8 and no cross reaction with BTV1-7, BTV9-24, Ibaraki virus, Chuzanvirus and Akabane virus. Western blot showed that the two MAbs reacted with the recombinant VP2. Further antigen epitope identification for the MAbs with 96 prokaryotic expressed maltose-binding protein （MBP）-fused peptides covering the VP2 showed that 2G4 recognized the linear antigenic determinants of 281^LCRLLSTIGRK MCNTE^296 on the VP2 of BTV8. The study renders BTV8 serotype-specific detection method establishment and structural and functional analysis of VP2 of BTV8 for further development.

关 键 词：蓝舌病病毒8型 VP2蛋白 真核表达 单克隆抗体 抗原表位 

分 类 号：S852.65[农业科学—基础兽医学]