摘要
[目的]本试验旨在制备特异性鼠抗猪瘟病毒(CSFV)非结构蛋白NS3和NS5B的多克隆抗体。[方法]根据CSFV NS5B基因序列设计1对特异性引物,以CSFV石门株为模板PCR扩增NS5B基因,同时根据大肠杆菌对密码子的偏嗜性优化了NS3密码子,并将NS3和NS5B基因分别克隆至原核表达载体p Cold I,转化至大肠杆菌Rossetta 2(R2),经0. 1mmol·L-1IPTG诱导后进行融合蛋白的表达和纯化,常规免疫BALB/c小鼠制备抗血清。应用Western blot和间接免疫荧光技术鉴定了多克隆抗体的特异性。[结果]Western blot结果显示,该抗血清不仅能识别原核和真核表达的NS3和NS5B蛋白,也能识别细胞中感染的CSFV并产生特异性条带;间接免疫荧光检测结果表明,2种抗血清除均能与胞浆中的CSFV粒子发生反应外,也能识别宿主细胞中真核表达的NS3和NS5B蛋白。[结论]本试验成功表达了NS3和NS5B,且用其制备的抗血清具有生物学功能,为深入探究猪瘟病毒致病机制奠定了基础。
[Objectives]The paper aims to prepare polyclonal antibody against non-structural protein 3(NS3)and non-structural protein 5B(NS5B)of classical swine fever virus(CSFV).[Methods]The NS 5 B gene was amplified from RNA of the CSFV Shimen strain using a pair of specific primers by RT-PCR.The NS 3 gene was codon-optimized according to the preference of Escherichia coli(E.coli).NS 3 and NS 5 B were cloned into prokaryotic expression vector pColdI,respectively,and transformed into E.coli R2(Rossetta 2).Fusion protein was expressed and purified after induced by 0.1 mmol·L^-1 IPTG,then polyclonal antibody was prepared by immunizing BALB/c mice with purified NS3 and NS5B protein.The specificity of polyclonal antibody was identified by Western blot and indirect immunofluorescence assay.[Results]Western blot results showed that antiserum could not only recognize prokaryotic and eukaryotic NS3 or NS5B protein,but also recognize CSFV,which caused specific bands;indirect immunofluorescence results showed that both antisera could react with CSFV particles in the cytoplasm and recognize the eukaryotic NS3 or NS5B protein in host cells.[Conclusions]The results showed that NS3 and NS5B protein were expressed successfully and the prepared antiserum had biological function.This study laid a foundation for further research of the role of NS3 and NS5B protein in the pathogenesis of CSFV.
作者
侯金秀
郑阳
李照耀
胡峰
陈熊男
张云娜
周斌
HOU Jinxiu;ZHENG Yang;LI Zhaoyao;HU Feng;CHEN Xiongnan;ZHANG Yunna;ZHOU Bin(College of Veterinary Medicine,Nanjing Agricultural University,Nanjing 210095,China;School Hospital,Nanjing Agricultural University,Nanjing 210095,China)
出处
《南京农业大学学报》
CAS
CSCD
北大核心
2018年第6期1093-1099,共7页
Journal of Nanjing Agricultural University
基金
国家重点研发计划项目(2016YFD0500701-3)
国家自然科学基金项目(31572554)
关键词
猪瘟病毒
非结构蛋白3(NS3)
NS5B
原核表达
多克隆抗体
鉴定
classical swine fever virus
non-structural protein 3(NS3)
non-structural protein 5B(NS5B)
prokaryotic expression
polyclonal antibody
identification
