A组猪轮状病毒NSP3蛋白单克隆抗体的制备及抗原表位鉴定  

Preparation of monoclonal antibody against NSP3 protein of group A porcine rotavirus and identification of antigenic epitopes

作　　者：谷凤丽[1] 石达[2] 时洪艳[2] 陈建飞[2] 张鑫[2] 袁婧[2] 徐天[2] 董慧[3] 冯力[2] 

机构地区：[1]东北农业大学生命科学学院,黑龙江哈尔滨150001 [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/猪消化道团队,黑龙江哈尔滨150001 [3]沈阳农业大学畜牧兽医学院,辽宁沈阳110866

出　　处：《中国预防兽医学报》2015年第4期295-298,共4页Chinese Journal of Preventive Veterinary Medicine

基　　金：黑龙江省高等学校科技创新团队项目(2011TD001)

摘　　要：为制备A组猪轮状病毒(Po RV)非结构蛋白NSP3的单克隆抗体(MAb)及鉴定其抗原表位,本研究将原核表达、纯化的重组NSP3蛋白(r NSP3)免疫BALB/c小鼠,取免疫小鼠的脾细胞与SP2/0细胞进行融合,经间接ELISA法筛选,得到一株能够稳定分泌抗Po RV(G9)的杂交瘤细胞株(5B8)。MAb鉴定结果显示其抗体亚类为Ig G1型;MAb细胞培养上清液效价为1∶103,western blot及间接免疫荧光结果表明该株MAb能够与Po RV反应。对NSP3蛋白进行截短表达,采用western blot试验确定所获得的MAb识别的抗原表位区域序列为290QDYD RTFL297。该MAb的制备为进一步研究NSP3蛋白的结构和功能奠定了良好的基础。To prepare the monoclonal antibody （MAb） against NSP3 of porcine group A rotavirus （PoRV） and identify epitope in NSP3, the NSP3 gene was cloned to pGEX-6P-1 vector and the recombinant NSP3 was expressed in E.coli as antigen and immune to the mice. Then, one hybridoma stably secreting MAb against NSP3 protein was prepared by fusing SP2/0 cells with spleen cells from BALB/c mice, and identified by indirect ELISA. The MAb belonged to IgG1 subtype. The titer in cell culture medium of the hybridomas reach to 1：10^3. In addition, the MAb was positively reacted with authentic NSP3 protein of PoRV verified by the western blot and indirect irnmunofluorescence assay. To identify the antigenic epitope against this MAb, a series of truncated NSP3 protein were expressed in E.coli and detected with the MAb by western blot method. The linear epitope sequence of ^290 QDYDRTFL^297 in NSP3 protein was identified, Those results provided a basis for the further study on the structure and function of the NSP3.

关 键 词：A组猪轮状病毒 NSP3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]