口蹄疫病毒3C蛋白多克隆抗体的制备与鉴定  

The preparation and identification of polyclonal antibody against FMDV 3C protein

作　　者：陈飞 吴香菊 齐静[2,3] 成子强[1] 杜以军[2,3] CHEN Fei;WU Xiangju;QI Jing;CHENG Ziqiang;DU Yijun(College of Veterinary Medicine,Shandong Agricultural University,Tai'an,271018,China;Shandong Province Key Laboratory of Animal Disease Control and Breeding,Jinan 250100,China;Institute of Animal Science and Veterinary Medicine,Shandong Academy of Agricultural Sciences,Jinan 250100,China)

机构地区：[1]山东农业大学动物科技学院,山东泰安271000 [2]山东省畜禽疫病防治与繁育重点实验室,山东济南250100 [3]山东省农业科学院畜牧兽医研究所,山东济南250100

出　　处：《畜牧与兽医》2019年第1期71-75,共5页Animal Husbandry & Veterinary Medicine

基　　金：国家重点研发计划课题(2016YFD0501505);泰山学者青年专家(tsqn20161057);山东省农业科学院农业科技创新工程(CXGC2016C07)

摘　　要：扩增口蹄疫病毒(FMDV) 3C蛋白编码基因,将其克隆到原核表达载体,并转化至大肠杆菌BL21中进行诱导表达; 3C重组蛋白纯化后免疫新西兰大白兔,制备多克隆抗体,通过ELISA和Western blot检测多克隆抗体的效价和特异性,并将该抗体通过间接免疫荧光(IFA)和Western blot应用于3C的检测。ELISA结果显示,制备的3C蛋白多克隆抗体效价达1∶256 000; IFA结果显示,该抗体能够检测到3C蛋白在细胞中的定位; Western blot结果显示,该多克隆抗体能够检测到在细胞中过表达的3C蛋白。本研究制备的FMDV 3C蛋白多克隆抗体为FMDV及其3C蛋白的相关研究提供了重要的物质基础。In this study,the FMDV 3C gene was amplified and cloned into a p ET-28 a vector to construct the prokaryotic expression plasmid p ET-28a-3C,which was further transformed into the Escherichia coli BL21 to induce expression. We used Ni-NTA agarose affinity chromatography and Millipore ultrafiltration tube to purify the 3C fusion protein. Then,New Zealand white rabbits were immunized with the purified recombinant protein to produce polyclonal antibodies. ELISA was used to detect the titer and Western blotting was used to analyze the specificity of the polyclonal antibody. The polyclonal antibody was further applied to detect the eukaryotic expression of the 3C protein by IFA and Western blotting. The results of ELISA showed that the titer of the 3C protein polyclonal antibody was as high as 1: 256 000. The polyclonal antibody detected the localization of the 3C protein in cells by IFA. The over-expressed 3C protein in cells could also be detected by Western blotting using the polyclonal antibody. The successful preparation of FMDV 3C polyclonal antibody here provided an important test tool for relevant study of FMDV and its 3C protein.

关 键 词：FMDV 3C蛋白 原核表达 蛋白纯化 多克隆抗体 

分 类 号：S852.6[农业科学—基础兽医学]