猪德尔塔冠状病毒N蛋白单克隆抗体2D7的制备与生物学特性鉴定  

Preparation and Characterization of Monoclonal Antibody 2D7 Against Porcine Deltacoronavirus N Protein

作　　者：张宁 赵平 刘思雨 赵硕 陈忠伟 何颖 欧阳康[3] 卢冰霞 蒋家霞 郭旋 林昌华 赵武 黄伟坚[3] 秦毅斌 赵凌[1] ZHANG Ning;ZHAO Ping;LIU Siyu;ZHAO Shuo;CHEN Zhongwei;HE Ying;OUYANG Kang;LU Bingxia;JIANG Jiaxia;GUO Xuan;LIN Changhua;ZHAO Wu;HUANG Weijian;QIN Yibin;ZHAO Ling(State Key Laboratory of Agricultural Microbiology,Huazhong Agricultural University,Wuhan 430070,China;Guangxi Veterinary Research Institute/Guangxi Key Laboratory of Veterinary Biotechnology/Key Laboratory of China(Guangxi)-ASEAN Cross-border Animal Disease Prevention and Control,Ministry of Agriculture and Rural Affairs of the People’s Republic of China,Nanning 530001,China;College of Animal Science and Technology,Guangxi University,Nanning 530005,China;Guangxi Vocational&Technical college,Nanning 530226,China;Guangxi Nongken Yongxin Animal Husbandry Group Jinguang Co.,Ltd.,Nanning,530042,China;Guangxi Nongken Yongxin Animal Husbandry Group Xijiang Co.,Ltd.,Guigang 537100,China;Guangxi Guiken Animal Husbandry Co.,Ltd.,Nanning 530022,China)

机构地区：[1]华中农业大学农业微生物学国家重点实验室,武汉430070 [2]广西壮族自治区兽医研究所/广西兽医生物技术重点实验室/农业农村部中国(广西)-东盟跨境动物疫病防控重点实验室,南宁530001 [3]广西大学动物科学技术学院,南宁530005 [4]广西职业技术学院,南宁530226 [5]广西农垦永新畜牧集团金光有限公司,南宁530042 [6]广西农垦永新畜牧集团西江有限公司,贵港537100 [7]广西桂垦牧业有限公司,南宁530022

出　　处：《中国动物传染病学报》2024年第5期115-125,共11页Chinese Journal of Animal Infectious Diseases

基　　金：南宁市优秀青年科技人才项目(RC20190102);广西农业科技自筹经费项目(Z202225);广西兽医生物技术重点实验室开放基金(22-035-32-B-01);桂职院[2022]133号221103。

摘　　要：为获得猪德尔塔冠状病毒(Porcine deltacoronavirus,PDCoV)N蛋白的特异性单克隆抗体(Monoclonal antibody,mAb),并鉴定其生物学特性,本研究利用RT-PCR方法扩增PDCoV-N基因,构建表达质粒,转化BL21感受态细胞获得重组大肠杆菌,诱导表达后获得PDCoV-N重组蛋白,将其进行纯化后再免疫小鼠,制备针对PDCoV-N蛋白的mAb,并进行了抗体的效价测定、免疫荧光(IFA)、Western blot、亚类的鉴定、mAb特异性的鉴定。结果,成功构建pColdⅡ-PDCoV-N重组原核表达质粒,SDS-PAGE显示重组N蛋白成功表达,分子质量约为38 kDa;经小鼠免疫、细胞融合、亚克隆筛选获得1株分泌PDCoV-N蛋白抗体的阳性杂交瘤细胞株;制备的腹水经间接ELISA方法测定抗体的效价为2.048×10^(6)。IFA、Western blot试验表明该mAb可以与PDCoV-N蛋白及全病毒特异性结合,亚类鉴定其亚型为IgG1,轻链为κ链。该mAb只与PDCoV反应,而与其他猪肠道病毒无交叉反应,表明本研究制备的mAb具有良好的特异性。本研究成功制备了PDCoV-N蛋白的mAb,为进一步研究PDCoV的生物学特性和诊断试剂的开发提供技术支持。To obtain monoclonal antibody(mAb)specific to porcine Delta-coronavirus(PDCoV)N protein and characterise its biological properties,the PDCoV-N gene was cloned the recombinant vector pColdⅡ-PDCOV-N was constructed for expression in E.coli.The recombinant PDCoV-N protein was purifi ed and used to immunize mice.As a result,a positive hybridoma cell line secreting specific antibodies against PDCoV-N protein was obtained through cell fusion,screening and cloning.The mAb was characterized through IFA,Western blot,subclass identifi cation,reactivity and titration.The showed that the recombinant N protein was expressed successesfully with the molecular weight of about 38 kDa as visualized in SDS-PAGE.The ELISA method for screening the mAb 2D7 utilized the His tag protein at 4μg/mL and antibody dilution at 1:200.The mAb 2D7 reacted with the recombinant N protein and intact PDCoV in IFA and Western blot.The subtype of the MAb 2D7 was IgG1 and light chain wasκchain.The ascitic fl uid of the mAb 2D7 had an ELISA titer at 2.048×10^(6)and protein concentration at 2.36 mg/mL.In summary,the mAb 2D7againstPDCoV-N protein was prepared in this study,which provided technical support for the further study of biological function and development of diagnostic methods of PDCoV.

关 键 词：猪德尔塔冠状病毒 N蛋白 单克隆抗体 

分 类 号：S852.651[农业科学—基础兽医学]