传染性支气管炎病毒N蛋白的原核表达及其多克隆抗体的制备与鉴定  

Preparation of Polyclonal Antibodies Against Recombinant Nucleocapsid Protein of Infectious Bronchitis Virus

作　　者：刘清艳 王欢 丁铲[2,3] 钱琨 廖瑛 方守国[1] LIU Qingyan;WANG Huan;DING Chan;QIAN Kun;LIAO Ying;FANG Shouguo(College of Animal Science,Yangtze University,Jingzhou 434025,China;Shanghai Veterinary Research Ins titut e,CAAS,Shanghai 200241,China;Jiangsu Key Laboratory of Zoonosis/Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses,Yangzhou University,Yangzhou 225000,China)

机构地区：[1]长江大学动物科学学院,荆州434025 [2]中国农业科学院上海兽医研究所,上海200241 [3]江苏省人兽共患病学重点实验室、江苏高校动物重要疫病与人兽共患病防控协同创新中心,扬州225000

出　　处：《中国动物传染病学报》2024年第4期179-187,共9页Chinese Journal of Animal Infectious Diseases

基　　金：国家自然科学基金面上项目(32172834);科技部十四五揭榜挂帅项目(2021YFD1801104);江苏省人兽共患病学重点实验室资助项目(R2007)。

摘　　要：本研究以IBV Beaudette株基因组为模板获得N基因片段,构建重组原核表达质粒pET-32a-His-IBV-N,在大肠杆菌BL21(DE3)诱导表达,纯化后获得带有His标签的N重组蛋白。将其与等体积的弗氏完全佐剂混匀乳化后免疫BALB/c小鼠3次,收集血清,得到鼠源抗N蛋白多克隆抗体。利用间接免疫荧光试验和Western blot鉴定多克隆抗体的特异性。结果显示,N蛋白多克隆抗体能特异性识别转染表达的Flag-N蛋白和IBV感染表达的N蛋白,说明此抗体能识别N蛋白的线性表位和空间表位,可应用于Western blot和间接免疫荧光实验。本研究为进一步研究N蛋白的功能和IB的诊断提供了技术支撑。The objective of this study was to prepare the murine-derived polyclonal antibodies(pAbs)against infectious bronchitis virus(IBV)N protein.The N gene was amplified by RT-PCR using IBV Beaudette genome as template and the amplified fragment was used for construction of the recombinant prokaryotic expression plasmid pET-32a-His-IBV-N.This plasmid was transformed into E.coli BL21(DE3)to express His-tagged recombinant protein N.BALB/c mice were immunized three times with the purified His-N protein emulsified in the same volume of Freund's complete adjuvant and the serum samples were collected for the following study.The specificity of the anti-N pAbs was examined in indirect immunofluorescence assay(IFA)and Western blot.The results showed that the murine anti-N pAbs specifically recognized both plasmid expressed N protein and virus expressed N protein.The availability of anti-N pAbs provided the tool for study of the biological function of N protein and offered the reagent for IB diagnosis.

关 键 词：传染性支气管炎病毒 N蛋白 多克隆抗体 

分 类 号：S852.65[农业科学—基础兽医学]