%0 Journal Article
%T 猪瘟病毒非结构蛋白NS3和NS5B多克隆抗体的制备和鉴定
%A 侯金秀
%A 郑阳
%A 李照耀
%A 胡峰
%A 陈熊男
%A 张云娜
%A 周斌
%J 南京农业大学学报
%D 2018
%R 10.7685/jnau.201804005
%X [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pColdI，转化至大肠杆菌Rossetta 2（R2），经0.1 mmol？L-1 IPTG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。</br>[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 NS5B gene was amplified from RNA of the CSFV Shimen strain using a pair of specific primers by RT-PCR. The NS3 gene was codon-optimized according to the preference of Escherichia coli (E. coli). NS3 and NS5B 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-1IPTG, 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
%K 猪瘟病毒
%K 非结构蛋白3(NS3)
%K NS5B
%K 原核表达
%K 多克隆抗体
%K 鉴定&lt
%K /br&gt
%K classical swine fever virus
%K non-structural protein 3(NS3)
%K non-structural protein 5B(NS5B)
%K prokaryotic expression
%K polyclonal antibody
%K identification
%U http://nauxb.njau.edu.cn/oa/darticle.aspx?type=view&id=201806015