口蹄疫病毒3B蛋白的表达纯化及其多克隆抗体的制备  

Expression and purification of the foot-and-mouth disease virus 3B protein and preparation of its polyclonal antibody

作　　者：辛旭 吴香菊[2,3] 齐静 王永志[4] 李相安[5] 杜正国 单虎 杜以军 XIN Xu;WU Xiangju;QI Jing;WANG Yongzhi;LI Xiang’an;DU Zhengguo;SHAN Hu;DU Yijun(College of Veterinary Medicine,Qingdao Agricultural University,Qingdao 266000,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;Jinan Vocational College of Nursing,Jinan 250100,China;Haidu College of Qingdao Agricultural University,Laiyang 265200,China;Yaodianzi Animal Husbandry and Veterinary Work Station,Animal Husbandry Bureau of Yishui County,Yishui 276400,China)

机构地区：[1]青岛农业大学动物医学院,山东青岛266000 [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 [4]济南护理职业学院,山东济南250100 [5]青岛农业大学海都学院,山东莱阳265200 [6]沂水县畜牧局姚店子畜牧兽医工作站,山东沂水276400

出　　处：《畜牧与兽医》2021年第3期109-114,共6页Animal Husbandry & Veterinary Medicine

基　　金：国家重点研发计划(2016YFD0501505);山东省高等学校优势学科创新团队培育计划(ZR2017MC002)。

摘　　要：为了深入研究口蹄疫病毒3B蛋白的结构及功能,本研究原核表达了3B蛋白并制备了针对3B蛋白的多克隆抗体。试验采用PCR方法扩增了口蹄疫病毒3B蛋白基因,并将其克隆到pET28a载体上,构建了重组质粒pET28a-3B,将其转化到大肠杆菌BL21感受态细胞中进行诱导表达。经过Ni-NTA琼脂糖亲和层析法初步纯化及Millipore超滤浓缩进一步纯化,将其免疫试验动物新西兰大白兔后制备出了抗3B蛋白的多克隆抗体。通过ELISA检测了该多抗的效价,Western blot试验检测了其反应性,通过间接免疫荧光(IFA)和Western blot试验检测了该多抗的应用性。结果表明:该抗体的效价为1∶512000,反应性良好;IFA试验和Western blot试验中可以检测到过表达后定位于细胞核和细胞质中的3B蛋白,也可以检测到在真核细胞中过表达的3B蛋白。说明本研究制备的口蹄疫病毒3B蛋白多克隆抗体可以作为开展该病毒相关研究的重要材料。In order to further study the structure and function of the 3 B protein of FMDV,we expressed the protein in the prokaryotic expression system in this study and prepared a polyclonal antibody against the protein.The PCR method was used to amplify the gene of the FMDV 3 B protein which was then cloned into a pET28 a vector.Recombinant plasmid pET28 a-3 B was constructed,and it was transformed into an E.coli BL21 strain for induced expression.Ni-NTA agarose affinity chromatography was used for preliminary purification,and Millipore ultrafiltration tubes were used for further ultrafiltration and concentration.The purified 3 B protein was immunized into New Zealand white rabbits to prepare a polyclonal antibody.The titer of the polyclonal antibody was detected by ELISA,and the reactivity of the polyclonal antibody was detected by Western blot,and finally the applicability of the polyclonal antibody was tested by IFA and Western blot.The results showed that the titer of the antibody was 1∶512000,and its reactivity was good.IFA and Western blot tests detected the 3 B protein located in the nucleus and cytoplasm after overexpressing,and also the 3 B protein after overexpressing in the eukaryotic cells,all of which suggested that the polyclonal antibody against the FMDV 3 B protein prepared in this study might be used as an important test tool for related research on FMDV.

关 键 词：口蹄疫病毒 3B蛋白 原核表达 蛋白纯化 多克隆抗体 

分 类 号：S855[农业科学—临床兽医学]