猪δ冠状病毒(PDCoV)N蛋白的原核表达及多克隆抗体制备  

Prokaryotic Expression and Polyclonal Antibody Preparation of Porcine Deltacoronavirus(PDCoV)N Protein

作　　者：陈汭 付嘉钰 刘浩宇 李成 赵玉佳 胡靖飞 瞿欢 曹三杰 文心田[1,2,3] 文翼平 赵勤[1,2,3] 伍锐 黄小波[1,2,3] CHEN Rui;FU Jia-yu;LIU Hao-yu;LI Cheng;ZHAO Yu-jia;HU Jing-fei;QU Huan;CAO San-jie;WEN Xin-tian;WEN Yi-ping;ZHAO Qin;WU Rui;HUANG Xiao-bo(Research Center for Swine Disease,College of Veterinary Medicine,Sichuan Agricultural University,Chengdu 611130;National Teaching and Experiment Center of Animal,Sichuan Agricultural University,Chengdu 611130;Sichuan Science-observation Experimental Station of Veterinary Drugs and Veterinary Diagnostic Technology,Ministry of Agriculture,Chengdu 611130)

机构地区：[1]四川农业大学动物医学院猪病研究中心,成都611130 [2]四川农业大学国家级动物类实验教学示范中心,成都611130 [3]农业部兽用药物与兽医诊断技术四川科学观测实验站,成都611130

出　　处：《生物技术通报》2020年第8期104-110,共7页Biotechnology Bulletin

基　　金：国家重点研发计划-猪重要疫病的诊断与检测新技术研究(2016YFD0500700);四川省科技计划资助(2020YFS0011);农业农村部农业重大技术协同推广计划试点项目;动物疫病共检测服务平台建设(D171100002117002)。

摘　　要：旨在表达和纯化猪δ冠状病毒(PDCoV)N蛋白并制备该蛋白的多克隆抗体。以RT-PCR扩增PDCoV N基因并与表达载体pET-28a构建重组质粒,转化Transetta(DE3)菌株诱导表达,SDS-PAGE鉴定融合蛋白表达,以纯化的N蛋白免疫家兔制备多克隆抗体,Western blot验证兔抗血清特异性,间接ELISA测定抗血清效价。利用间接免疫荧光试验(IFA)、免疫荧光试验(IF)、流式细胞术(FCM)鉴定其诊断应用价值。重组N蛋白为可溶性表达,大小约为44 kD,制备的兔抗N蛋白抗体效价可达1∶204800。IFA与FCM试验证实该抗体能与PDCoV特异性结合,与PEDV及TGEV无交叉反应,IF试验表明该抗体可用于检测小肠组织中的PDCoV。This work aims to express and purify the porcine deltacoronavirus(PDCoV)N protein and to prepare its polyclonal antibody.The PDCoV N gene was amplified by RT-PCR and the recombinant prokaryotic expression vector pET28-N was transformed into Escherichia coli Transetta(DE3),from which the fusion protein was identified by SDS-PAGE.The purified N protein was used to inoculate rabbit and the polyclonal antibody was prepared.The specificity of the serum antibody was determined by Western blot and the serum antibody titer was detected by indirect ELISA.The diagnostic application potential of the polyclonal antibody was evaluated by using indirect immunofluorescence assay(IFA),immunofluorescence staining(IF)and flow cytometry(FCM).Recombinant N protein was soluble with a molecular weight of 44 kD.The titer of the antibody was about 1∶204800.IFA and FCM tests confirmed that the antibody specifically bound to PDCoV and had no cross-reactivity with PEDV and TGEV.IF tests showed that the polyclonal antibody could be used to detect PDCoV in small intestine tissue.

关 键 词：猪δ冠状病毒 N蛋白 原核表达 多克隆抗体 

分 类 号：S852.651[农业科学—基础兽医学]