牛病毒性腹泻病毒衣壳蛋白原核表达及多克隆抗体的制备  

Prokaryotic expression of caspid protein of bovine viral diarrhea virus and polyclonal antibody preparation

作　　者：张忠辉 高闪电[1] 独军政[1] 田占成[1] 王建东 殷宏[1,3] ZHANG Zhonghui;GAO Shandian;DU Junzheng;TIAN Zhancheng;WANG Jiandong;YIN Hong(State Key Laboratory of Veterinary Etiological Biology,Lanzhou VeterinaryResearch Institute,Chinese Academy of Agricultural Sciences,Lanzhou 730046,China;Institute of Animal Science,Ningxia Academy of Agriculture and Forestry Sciences,Yinchuan 750002,China;Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses,Yangzhou 225009,China)

机构地区：[1]中国农业科学院兰州兽医研究所家畜疫病病原生物学国家重点实验室,甘肃兰州730046 [2]宁夏农林科学院动物科学研究所,宁夏银川750002 [3]江苏省动物重要疫病与人兽共患病防控协同创新中心,江苏扬州225009

出　　处：《畜牧与兽医》2020年第7期78-82,共5页Animal Husbandry & Veterinary Medicine

基　　金：国家重点研发计划项目(2017YFD0500904);宁夏农林科学院动物科学研究所对外合作项目(DW-X-2018020)。

摘　　要：试验旨在通过原核表达获得牛病毒性腹泻病毒(BVDV)衣壳蛋白(C),以制备抗C蛋白多克隆抗体。根据BVDV C蛋白的编码序列设计一对特异性引物,利用PCR扩增编码BVDV C蛋白片段,定向插pPET-42a载体中,转化大肠杆菌BL21 Rosetta (DE3) pLysS感受态细胞,筛选重组阳性菌株,IPTG诱导表达,金属离子层析法纯化得到分子质量为45 kDa的重组C蛋白。利用纯化的重组C蛋白免疫新西兰大白兔制备多克隆抗体,间接ELISA检测多克隆抗体效价达到1∶25 600。经间接免疫荧光证实制备的多克隆抗体可与细胞中的BVDV抗原发生反应,具有良好的反应性。本研究成功制备了BVDV重组C蛋白的兔源多克隆抗体,为BVDV衣壳蛋白功能的研究奠定了基础。The experiment was aimed to obtain a bovine viral diarrhea virus(BVDV) capsid(C) protein by prokaryotic expression and to prepare an anti-C protein polyclonal antibody. A pair of specific primers was designed according to the coding sequence of the BVDV C protein. The BVDV C protein coding fragment was amplified by PCR and then inserted into the PET-42 a vector, followed by transformation into BL21 Rosetta(DE3) pLysS competent cells. The recombinant strain was screened and induced with IPTG. The recombinant C protein with a molecular weight of 45 kDa was purified by metal ion chromatography. New Zealand white rabbits were immunized with the recombinant C protein. The polyclonal antibody was obtained after three immunizations. The titer of the polyclonal antibody was determined to be over 1:25600 by indirect ELISA. The reactivity of the polyclonal antibody with the BVDV antigen in the cell culture was confirmed by indirect immunofluorescence. The rabbit-derived polyclonal antibodies of the BVDV C protein were successfully prepared, which laid a foundation for the further study of the C protein function.

关 键 词：牛病毒性腹泻病毒 衣壳蛋白 表达 多克隆抗体 

分 类 号：S852.65[农业科学—基础兽医学]