一株抗猪脑心肌炎病毒VP2蛋白单克隆抗体的制备及抗原表位鉴定  

Preparation of monoclonal antibodies against encephalomyocarditis virus VP2 protein and identification the B-cell epitope on VP2

作　　者：张泉[1,2] 张元峰 黄丽[2] 王胜男[2] 蔡雪辉[2] 熊涛[1] 翁长江[2] 

机构地区：[1]长江大学生命科学学院,湖北荆州434025 [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/动物病原监测与流行病学研究创新团队,黑龙江哈尔滨150001

出　　处：《中国预防兽医学报》2015年第7期553-556,共4页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家自然科学基金(31300139);国家自然科学基金(31172333)

摘　　要：为制备抗猪脑心肌炎病毒(EMCV)HB10株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(r VP2),纯化后将其免疫BALB/c小鼠,取免疫的小鼠脾细胞与SP2/0细胞融合,通过间接ELISA方法筛选出一株稳定分泌抗VP2 MAb(1D3)。通过间接免疫荧光试验和western blot对该MAb的免疫活性和特异性进行鉴定,结果表明1D3 MAb仅能够特异性与EMCV全病毒反应。此外,通过构建截短的VP2蛋白重组质粒,采用western blot的方法对VP2蛋白抗原表位进行鉴定,初步确定MAb 1D3识别的线性表位区域为89DGGVFGAALRRH100。本实验结果为进一步研究VP2蛋白的功能及建立EMCV检测方法奠定了基础。To identify the epitope on encephalomyocarditis virus（EMCV） VP2 protein, the monoclonal antibody（MAb）against VP2 protein was prepared by fusion of SP2/0 cells with the splenic cells from immunized BALB/c mice with purified recombinant VP2 protein expressed in E.coli. A positive hybridoma cell line stably secreting MAb, named 1D3, was selected by 3cycles of limited dilutions and identified by indirect ELISA coated with the purified recombinant VP2. Immunological activity and specificity of the MAb was identified by indirect immunofluorescence assay and western blot. The result showed that the MAb was able to react with recombinant VP2. In addition, western blot analysis showed that the MAb was capable of reacting with VP2-2（aa65-aa128） fragment expressed in E.coli. Furthermore, the results displayed that the MAb recognized the linear epitope at89DGGVFGAALRRH100. Those results provide a basis for the further study on the function of EMCV VP2 and development of diagnostic method.

关 键 词：猪脑心肌炎病毒 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]