盖塔病毒E2蛋白单克隆抗体的制备及其抗原表位鉴定  

Preparation of monoclonal antibody against E2 protein of Getah virus and identification of its epitopes

作　　者：孟慧 鹿田原 周景业 牟春晓 陈振海 MENG Hui;LU Tianyuan;ZHOU Jingye;MOU Chunxiao;CHEN Zhenhai(College pf Veterinary Medicine,Yangzhou University,Yangzhou 225009,China)

机构地区：[1]扬州大学兽医学院,江苏扬州225009

出　　处：《扬州大学学报（农业与生命科学版）》2023年第3期51-58,共8页Journal of Yangzhou University：Agricultural and Life Science Edition

基　　金：国家重点研发计划项目(2021YFD1800104);江苏高校优势学科建设工程项目(PAPD);江苏高校动物重要疫病与人兽共患病防控协同创新中心开放课题(201810)。

摘　　要：为制备盖塔病毒(GETV)E2蛋白单克隆抗体,通过大肠埃希菌表达系统表达重组E2蛋白,经过镍柱吸附、切胶纯化,获得E2蛋白,将其作为免疫原免疫BALB/c小鼠,3次免疫后加强免疫,细胞融合,通过间接免疫荧光筛选阳性杂交瘤细胞,最终筛选出1株针对GETV E2蛋白的阳性杂交瘤细胞9E8,将该杂交瘤细胞注射入小鼠腹腔内生产腹水。经间接免疫荧光试验和蛋白免疫印迹试验鉴定,该单抗可与GETV发生特异性反应。经IP试验证明,该单抗可识别天然结构的GETVE2蛋白。对E2单抗进行抗原表位鉴定,确定其所识别的多肽序列为66KIRYIAGHD74。与GenBank上登录的其他GETV毒株序列对比,发现该段序列高度保守。综上,E2单抗9E8免疫学反应特性良好,为研究该病毒提供了有效的免疫学检测工具。In order to prepare the McAbs against Getah virus(GETV)E2 protein,we expressed recombinant E2 protein by E.coli expression system.The E2 protein was collected for purification by Ni^(2+)affinity chromatography and gelslicing to immune BALB/c mice as immunogen,after three times immunization and strengthen immunity,cell fusion,the positive hybridoma cells were screened by indirect immunofluorescence.Finally,a hybridoma cell strain 9E8,which was positive for GETV E2 protein,was screened out,and it was injected into the abdominal cavity of mice to produce ascites.Indirect immunofluorescence assay and Western blot identification showed that the monoclonal antibody could react specifically with GETV.IP test showed that the monoclonal antibody could recognize the natural structure of GETV E2 protein.Antigenic epitopes of E2 monoclonal antibody were identified and the recognized polypeptide sequence was 66KIRYIAGHD74.Compared with other GETV strains recorded in GenBank,this sequence was highly conserved.The results showed that E2 monoclonal antibody 9E8 had good immunological characteristics and provided an effective immunological detection tool for the study of the virus.

关 键 词：盖塔病毒 E2蛋白 单克隆抗体 抗原表位 

分 类 号：S852.65[农业科学—基础兽医学]