传染性支气管炎病毒非结构蛋白nsp15的体外表达及其单克隆抗体的制备  

Expression of the nonstructural protein 15 of infectious bronchitis virus and preparation of its monoclonal antibody

作　　者：李乐超[1] 石婷婷[1] 郎咸勇 牟小东[1] 王丹丹[1] 周继勇[1] 廖敏[1] 

机构地区：[1]浙江大学农业部动物病毒学重点实验室,浙江杭州310058

出　　处：《中国兽医科学》2016年第1期52-57,共6页Chinese Veterinary Science

基　　金：国家自然科学基金项目(31302104);现代农业产业技术体系岗位专家项目(CARS-41-11)

摘　　要：以实验室保存的肾型传染性支气管炎病毒(IBV)毒株SC021202作为模板,扩增IBV非结构蛋白nsp15基因后,成功构建了原核表达载体PET-28a(+)-nsp15,将其转化大肠杆菌,经IPTG诱导表达出约45ku的目的蛋白。纯化的nsp15蛋白可与IBV感染鸡血清发生良好反应。用纯化的重组nsp15蛋白免疫小鼠,制备单克隆抗体。经过3轮有限稀释和筛选阳性克隆,最终获得3株稳定分泌nsp15蛋白抗体的细胞株,分别为1A6、2B6和4D7。Western-blot分析显示,3株单克隆抗体都与nsp15蛋白有很好的特异性反应。IFA检测显示,nsp15单克隆抗体与IBV感染的细胞和nsp15基因转染的细胞均出现明显的反应。IBV nsp15单克隆抗体的制备为传染性支气管炎流行病学的监测、鉴别诊断及对其蛋白功能的进一步研究奠定了基础。Nonstructural protein 15（nsp15）gene of infectious bronchitis virus（IBV）was amplified by RT-PCR with cDNA of IBV SC021202 strain as template.The nsp15 gene was then cloned into prokaryotic expression vector pET-28a（＋）and transformed into Escherichia coli.Expression of recombinant Hisnsp15 protein in E.coli was observed under 1mmol/L IPTG induction for 6hours,which showed a good reaction with antiserum to IBV.For preparation of monoclonal antibody（MCAb）against the nsp15,BALB/c mice were immunized with the purified recombinant His-nsp15.After three times limiting dilution,three hybridomas cell strains（1A6,2B6 and 4D7）secreted antibodies and reacted with nsp15 by Western-blot analysis were obtained.The three McAbs could react with IBV infected cells and nsp15 transfected cells in IFA.The monoclonal antibodies against IBV nsp15 were successfully prepared,which might provide an efficient tool for IBV detection and further study of the functions of IBV nsp15.

关 键 词：传染性支气管炎病毒 非结构蛋白15 单克隆抗体 

分 类 号：S852.659.6[农业科学—基础兽医学]