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
李丹彤 1何庆 1王东亮 1喻蓓蕾 1袁倩 1刘伟姣 1杨毅 1王乃东1
作者信息
- 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
- 折叠
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.
Abstract
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 structur-al 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 mono-clonal 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 superna-tants(MAb)specifically recognizing S protein(2E5,4D5,5D10,2F7 and 5A9)were identified by Western blot and immunofluorescence assay(IFA).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 se-crete and specifically bind to PDCoV and S protein were prepared,which laid an important founda-tion for further research on the structure and function of S protein,the development 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Key words
porcine deltacoronavirus/S protein/baculovirus expression vector system/monoclonal antibody/neutralizing antibody引用本文复制引用
出版年
2024
NETL
NSTL
万方数据