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.
出处
《中国预防兽医学报》
CAS
CSCD
北大核心
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)
