【摘要】 为制备蓝舌病病毒(BTV)8型VP2蛋白的单克隆抗体(MAb)及鉴定其抗原表位,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3B7)。间接免疫荧光结果表明:2G4和3B7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。Western blot结果显示,2G4和3B7均能够识别BTV8及BTV8重组VP2蛋白。Ig亚类鉴定2G4和3B7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:MAb 2G4识别的抗原表位为281LCRLLSTIGRKMCNTE296。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。更多还原

【Abstract】 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 of281LCRLLSTIGRK MCNTE296on 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.更多还原