猪δ冠状病毒N蛋白单克隆抗体的制备及初步应用  被引量:1

Preparation and Preliminary Application of Monoclonal Antibodies Against Recombinant N Protein of Porcine Deltacoronavirus

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作  者:方谱县 张蕙畅 夏思进 江晓翠 孙倩倩 肖少波[1,2] 方六荣 FANG Puxian;ZHANG Huichang;XIA Sijin;JIANG Xiaocui;SUN Qianqian;XIAO Shaobo;FANG Liurong(State Key Laboratory of Agricultural Microbiology,College of Veterinary Medicine,Huazhong Agricultural University,Wuhan 430070,China;The Cooperative Innovation Center for Sustainable Pig Production,Wuhan 430070,China)

机构地区:[1]华中农业大学动物医学院农业微生物学国家重点实验室,武汉430070 [2]湖北省生猪健康养殖协同创新中心,武汉430070

出  处:《中国动物传染病学报》2023年第6期1-10,共10页Chinese Journal of Animal Infectious Diseases

基  金:国家自然科学基金(31902247)。

摘  要:为制备猪δ冠状病毒(PDCoV)N蛋白的特异性单克隆抗体,并初步应用于临床样品中PDCoV抗原的免疫组织化学(IHC)检测,本研究以原核表达的PDCo V N蛋白免疫BALB/c小鼠,取其脾细胞与骨髓瘤细胞进行融合,基于纯化的N蛋白建立的间接ELISA方法筛选获得稳定分泌N蛋白特异性单克隆抗体的杂交瘤细胞,并对单克隆抗体的免疫反应特异性、亚类进行系统鉴定。结果显示:通过3次亚克隆筛选获得8株可分泌PDCo V N蛋白单克隆抗体的杂交瘤细胞株。经IFA和Western blot分析,证实制备的单克隆抗体均与猪δ冠状病毒反应,且不与猪流行性腹泻病毒(PEDV)和猪传染性胃肠炎病毒(TGEV)反应,特异性良好。单克隆抗体亚类鉴定结果表明,1A11、3C6、1E7、1E12株单克隆抗体属Ig G1型,4F3、2A3株单克隆抗体为Ig G2a型,3E4单克隆抗体为Ig M型,8株单克隆抗体的轻链均为κ链。利用1A11单克隆抗体对PDCo V、PDEV、TGEV感染猪的空肠、回肠组织进行IHC检测,结果表明1A11能与感染PDCo V的组织发生特异性免疫反应,而不与感染PDEV和TGEV的组织发生反应,特异性良好。本研究制备的PDCo V N蛋白单克隆抗体可为PDCo V诊断方法的建立以及N蛋白的功能研究奠定基础。The objectives of this study were to prepare specific monoclonal antibodies(MAbs)against N protein of Porcine deltacoronavirus(PDCoV)and preliminarily apply them to detect PDCoV antigen in clinical samples in immunohistochemistry(IHC).BALB/c mice were immunized with purified recombinant N protein expressed by prokaryotic expression system followed by fusion of the harvested spleen cells and myeloma cells.The hybridomas stably secreting MAbs against N protein were selected in indirect ELISA using the purified N protein.The results showed that 8 monoclonal cell lines secreting MAbs against PDCoV N protein were obtained through screenings and subsequent three cycles of subclonings.All 8 MAbs reacted with PDCoV in IFA and Western blotting but not with Porcine epidemic diarrhea virus(PEDV)and Porcine transmissible gastroenteritis virus(TGEV).In addition,the heavy chains of MAbs 1A11,3C6,1E7 and 1E12 belonged to IgG1,4F3 and 2A3 to IgG2a and 3E4 to IgM.The light chains of all MAbs wereκ.Then the MAb 1A11 was used in IHC to examine the jejunum and ileum tissues infected with PDCoV,PDEV and TGEV.As a result,that the MAb 1A11 reacted specifically with PDCoV-infected tissues but not with PDEV and TGEV-infected tissues,suggesting its good specificity.The availability of MAbs against PDCoV N protein laid an important foundation for the development of PDCoV diagnostic methods and research of the function of N protein.

关 键 词:猪δ冠状病毒 N蛋白 原核表达系统 单克隆抗体 免疫组化 

分 类 号:S852.4[农业科学—基础兽医学]

 

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