猪流行性腹泻病毒nsp8蛋白的原核表达及其多克隆抗体的制备  

Prokaryotic expression of nonstructural protein 8 of porcine epidemic diarrhea virus and preparation of its polyclonal antibodies

作　　者：朱婷 洪润婧 徐雅雯 范红结[1] 蔺辉星[1] ZHU Ting;HONG Run-jing;XU Ya-wen;FAN Hong-jie;LIN Hui-xing(College of Veterinary Medicine,Nanjing Agricultural University,Nanjing 210095,China)

机构地区：[1]南京农业大学动物医学院,江苏南京210095

出　　处：《中国兽医科学》2023年第10期1279-1284,共6页Chinese Veterinary Science

基　　金：国家重点研发计划项目(2022YFD1800900);国家自然科学基金项目(31702278);大学生创新训练计划(SRT)项目(202217YX777,202317XX47);江苏高校优势学科建设工程专项资金项目(PAPD)。

摘　　要：复制/转录复合物(RTC)在冠状病毒复制过程中具有重要作用。猪流行性腹泻病毒(PEDV)的非结构蛋白8(nsp8)参与病毒RTC的形成。因此,可将nsp8作为RTC的检测标志之一。为获得PEDV nsp8蛋白并制备其多克隆抗体,本研究通过同源重组的方法将PEDV nsp8基因与质粒pET-28a(+)连接。将重组质粒pET-28a-nsp8转化至大肠杆菌BL21感受态细胞进行诱导表达。用镍柱纯化6His-nsp8蛋白并对其进行复性,然后用凝血酶酶切去除his标签,得到纯化的nsp8蛋白。SDS-PAGE结果显示,重组nsp8蛋白大小为27 ku。以纯化的nsp8蛋白免疫新西兰大白兔制备多克隆抗体。Western-blot结果表明,本研究制备的家兔抗nsp8多克隆抗体可与nsp8蛋白反应。用该多克隆抗体进行间接免疫荧光(IFA)试验,可在PEDV感染细胞的细胞核周围观察到明显的荧光,表明nsp8多克隆抗体可用于病毒RTC的检测。上述研究结果为冠状病毒RTC的形成机制及其功能研究奠定了基础。Replication/transcription complexes(RTC)play an important role in coronavirus replication.The nonstructural protein 8(nsp8)of porcine epidemic diarrhea virus(PEDV)is involved in the formation of RTC.Therefore,nsp8 can be used as one of the detection markers of RTC.In order to obtain the PEDV nsp8 protein and prepare the anti-nsp8 polyclonal antibody,the PEDV nsp8 gene was amplified and inserted into the pET-28a(+)plasmid by homologous recombination.Recombinant plasmid pET-28a-nsp8was transformed into E.coli BL21.Purify the 6His-nsp8 protein using a nickel column and renaturate it,then remove the 6His tag with thrombin digestion to obtain the purified nsp8 protein.SDS-PAGE showed that the weight of nsp8 was about 27 ku.Polyclonal antibodies were prepared by immunizing New Zealand white rabbits with purified nsp8 protein,and the results of Western-blot showed that the rabbit anti-nsp8 polyclonal antibody prepared in this study could react with nsp8 protein.Indirect immunofluorescence assays(IFA)showed that fluorescence could be observed around the nucleus of PEDV infected cells,indicating the anti-nsp8 polyclonal antibody can be used for the detection of viral RTC.The above results provide a basis for the research on the formation mechanism and function of coronavirus RTC.

关 键 词：猪流行性腹泻病毒 非结构蛋白8 多克隆抗体 复制/转录复合物 

分 类 号：S852.659.6[农业科学—基础兽医学]