蓝舌病病毒15型VP2蛋白单克隆抗体的制备及其抗原表位鉴定  

Preparation of the monoclonal antibody against VP2 protein of bluetongue virus serotype 15 and identification of B-cell epitope

作　　者：张沁[1] 孙恩成[1] 徐青元[1] 杨涛[1] 王海秀[1] 冯瑜菲[1,2] 李俊平[1] 吕爽[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 [2]东北农业大学动物医学学院,黑龙江哈尔滨150030

出　　处：《中国预防兽医学报》2015年第5期392-396,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(SKLVBP201415)

摘　　要：为制备抗蓝舌病病毒(BTV)15型VP2蛋白的单克隆抗体(MAb)及鉴定其抗原表位,本研究利用原核表达系统表达的重组VP2蛋白纯化后免疫BALB/c小鼠,将其脾细胞与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并纯化的重组VP2蛋白为包被抗原,通过间接ELISA筛选出一株稳定分泌抗BTV15VP2蛋白的MAb(2D6)。通过western blot和间接免疫荧光试验对该MAb进行特异性鉴定。结果显示,其仅能够识别BTV15,而不能识别其他血清型BTV以及同种属的中山病毒和茨城病毒,表明该MAb具有良好的反应特异性。利用肽扫描技术对该MAb针对的B细胞表位进行鉴定,确定该MAb识别的抗原表位为15IAPAIIK RYP24。本研究为BTV15特异性抗原检测方法的建立奠定了基础。To prepare the monoclonal antibody （MAb） against the VP2 protein of bluetongue virus （BTV） 15 serotype and identify the linear epitope, the SP2/0 cells were fused with the spleen cells of the BALB/c mouse which was immunized by purified recombinant VP2 protein （rVP2） expressed in E.coli. The hybridoma cells were screened by indirect ELISA coated with the purified rVP2 expressed from baculovirus expression system. One positive hybridoma cells line stably secreting antibody against VP2 of BTV15 was identified and verified by western blot. In addition, indirect immunofluorescence assay showed that the MAb reacted only with the BHK cells infected by BTV15, but did not to react with BTV1 to 14 and BTV16 to 24, Ibaraki virus and Chuzan virus. The linear epitope in VP2 of BTV15 was mapped by the pepscan method with the MAb, and found that the MAb recognized the sequence of sIAPAIIKRYP4 in the VP2. The preparation of the MAb and identification of linear epitope in VP2 of BTV15 lays the foundation for BTV1 5 serotype-specific detection method establishment.

关 键 词：蓝舌病病毒15型 VP2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]