摘要
Rep蛋白在番鸭细小病毒(Muscovy duck parvovirus,MDPV)感染中的作用尚不清楚。研究将MDPV的Rep1基因克隆入p ET-28a原核表达载体并转化BL21(DE3)大肠杆菌,在IPTG诱导下成功表达了分子量约为82 ku的目的蛋白,菌体分析表明目的蛋白主要以包涵体存在。将重组蛋白切胶免疫ICR小鼠,制备了针对Rep1蛋白的多克隆抗体。在间接免疫荧光试验中,Rep1多克隆抗体能与鹅胚成纤维细胞上增殖的MDPV特异性发生反应,免疫印迹试验则显示Rep1多克隆抗体能够在MDPV感染的GEF中识别出两条蛋白条带Rep1和Rep2。结果表明制备的Rep1多克隆抗体具有良好反应特异性,为深入研究Rep蛋白在MDPV感染中的作用奠定了基础。
The function of Rep proteins in the course of Muscovy duck parvovirus （MDPV）infection of host cells remains unclear. In this study, Repl gene was cloned into the prokaryotic expression vector pET-28a, resulting in the recombinant plasmid pET28-Rep1. The pET28-Rep1 plasmid was transformed into BL21 （DE3）E. coli for expression under induction of IPTG. The desired protein with a molecular mass of 82 ku was observed in SDS-PAGE analysis with the cell lysates, and was found to be expressed as inclusion body. The gel containing the desired protein band was sliced and homogenized, which was further used to immunize ICR mice for four times to produce polyclonal antisera against Repl protein. In the indirect immunofluorescence assay （IFA）, Rep1 antisera could specifically react with MDPV propagating in goose embryo fibroblast （GEF）. Immunoblotting revealed that the Rep1 antisera could recognize two proteins （Rep1 and Rep2）in GEF infected with MDPV. The results demonstrated that the prepared Rep1 antisera had a good reactivity with MDPV and provided a solid foundation for exploring the role of Rep proteins in MDPV infection of host cells in the future.
作者
王志仙
凌珏怡
贾婧宇
王建业
朱国强
WANG Zhixian;LING Jueyi;JIA Jingyu;WANG Jianye;ZHU Guoqiang(College of Veterinary Medicine, Yangzhou University, Yangzhou, Jiangsu 225009;Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, Jiangsu 225009)
出处
《中国家禽》
北大核心
2018年第6期15-18,共4页
China Poultry
基金
国家自然科学基金项目(31572511)
江苏高校优势学科建设工程资助项目
关键词
番鸭细小病毒
Rep1蛋白
原核表达
间接免疫荧光试验
Muscovy duck parvovirus
Rep1 protein
prokaryotic expression
indirect immunofluorescence assay
