猪丁型冠状病毒S蛋白在昆虫细胞中的表达及单克隆抗体的制备与鉴定  

Expression of porcine deltacoronavirus S protein in insect cells and preparation and identification of monoclonal antibody

作　　者：李丹彤 何庆 王东亮 喻蓓蕾 袁倩 刘伟姣 杨毅 王乃东 LI Dantong;HE Qing;WANG Dongliang;YU Beilei;YUAN Qian;LIU Weijiao;YANG Yi;WANG Naidong(R&D Center for Animal Reverse Vaccinology of Hunan Province,Hunan Provincial Key Laboratory of Protein Engineering in Animal Vaccines,College of Veterinary Medicine,Hunan Agricultural University,Changsha 410128,China)

机构地区：[1]湖南农业大学动物医学院兽用蛋白质工程疫苗湖南省重点实验室/兽用疫苗逆向创制湖南省工程研究中心,湖南长沙410128

出　　处：《中国兽医学报》2024年第11期2309-2315,共7页Chinese Journal of Veterinary Science

基　　金：湖南省技术攻关“揭榜挂帅”资助项目(2021NK1030);湖南省自然科学基金资助项目(2022JJ30298)

摘　　要：猪丁型冠状病毒(PDCoV)是近年来新发现的冠状病毒,可引起仔猪严重的腹泻、呕吐、脱水甚至死亡。S蛋白是PDCoV的重要结构蛋白,决定着病毒的宿主或组织嗜性,也是研制PDCoV疫苗和检测方法的重要靶点。为制备PDCoV S蛋白的单克隆抗体,本研究基于国内PDCoV流行毒株S蛋白胞外域序列,构建S蛋白重组质粒转化DH10Bac感受态细胞,经蓝白斑筛选及PCR鉴定重组Bacmid,通过昆虫-杆状病毒系统表达S蛋白。将获得的S蛋白免疫BALB/c小鼠,并以免疫后小鼠脾细胞与骨髓瘤细胞融合,通过间接ELISA方法和亚克隆筛选出稳定分泌抗体的阳性杂交瘤细胞。免疫印迹和间接免疫荧光试验(IFA)鉴定出5株(2E5、4D5、5D10、2F7和5A9)特异性识别S蛋白的杂交瘤细胞。中和试验表明,3株单克隆抗体(2E5、4D5和5D10)具有病毒中和作用。本研究成功表达了S蛋白及制备了5株能稳定分泌与PDCoV病毒和S蛋白特异性结合的单克隆抗体,为进一步研究S蛋白的结构与功能、PDCoV感染的检测方法开发、预防或治疗用制剂等研究奠定了重要基础。Porcine deltacoronavirus(PDCoV),a newly discovered virus in recent years,can cause severe diarrhea,vomiting,dehydration and even death in piglets.S protein is an important structural protein of PDCoV,which determines the host or tissue tropism of the virus,and is an important target for the development of PDCoV vaccines and detection methods.In order to prepare monoclonal antibody(MAb)against the S protein of PDCoV,the recombinant plasmid of S protein was constructed based on the extracellular domain sequence of S protein of PDCoV epidemic strain in China and transformed into DH10Bac competent cells.The recombinant bacmid was identified by blue-white spot screening and PCR.BALB/c mice were immunized with S protein,and the spleen cells of immunized mice were fused with myeloma cells.The positive hybridoma cells that secreted stable antibodies were screened by indirect ELISA and subcloning.Five hybridoma cell supernatants(MAb)specifically recognizing S protein(2E5,4D5,5D10,2F7 and 5A9)were identified by Western blot and immunofluorescence assay(IF A).Subsequently,the neutralization test showed that three of the monoclonal antibodies(2E5,4D5 and 5D10)could neutralize the virus to varying degrees.The S protein was successfully expressed and 5 monoclonal antibodies that can stably secrete and specifically bind to PDCoV and S protein were prepared,which laid an important foundation for further research on the structure and function of S protein,the developmenl of detection methods for PDCoV infection,and the prevention or treatment of PDCoV infection.

关 键 词：猪丁型冠状病毒 S蛋白 昆虫-杆状病毒表达 单克隆抗体 中和抗体 

分 类 号：S852.65[农业科学—基础兽医学]