非洲猪瘟病毒p72蛋白单克隆抗体的制备及鉴定  

Preparation and Characterization of Monoclonal Antibodies Against p72 Protein of African Swine Fever Virus

作　　者：王彩霞[1] 冯春燕[1] 于浩洋 宋晓晖[2] 仇松寅[1] 刘晓飞[1] 吴绍强[1] 林祥梅[1] WANG Caixia;FENG Chunyan;YU Haoyang;SONG Xiaohui;QIU Songyin;LIU Xiaofei;WU Shaoqiang;LIN Xiangmei(Animal Inspection and Quarantine Institute,Chinese Academy of Inspection and Quarantine,Beijing 100176,China;China Animal Disease Control Center,Beijing 102600,China)

机构地区：[1]中国检验检疫科学研究院动物检验与检疫研究所,北京100176 [2]中国动物疫病预防控制中心,北京102600

出　　处：《中国动物传染病学报》2023年第5期188-193,共6页Chinese Journal of Animal Infectious Diseases

基　　金：十三五国家重点研发计划项目(2016YFD0501100);中国检验检疫科学研究院基本科研业务经费(2020JK020)。

摘　　要：本研究将含有非洲猪瘟病毒p72全长基因序列的pCMV-p72重组质粒与B602L质粒共同转染HEK293F细胞,纯化获得重组p72-His蛋白,经SDS-PAGE和间接ELISA鉴定后,以该纯化蛋白免疫BALB/c小鼠,进行融合、筛选获得4株单克隆抗体。经亚型鉴定,2B2-1、34C10和22D3均为IgG1/κ型,32H5为IgG2b/κ型,4株单克隆抗体的腹水效价均高于6.4×104。免疫荧光检测结果显示4株单克隆抗体均能够特异性识别ASFV p72蛋白,亲和力试验显示4株单克隆抗体的亲和力由高到低依次为22D3>32H5>2B2-1=34C10,表位竞争试验也揭示这4株单克隆抗体可识别不同的抗原结合位点,且2B2-1和32H5能组成识别抗原的抗体对,34C10单克隆抗体具有竞争效应。所获得的4株杂交瘤细胞株均能够稳定分泌抗体,且分泌的抗体特异性高,亲和力强,有助于进一步研发ASFV血清学诊断方法。In order to obtain monoclonal antibodies(MAbs)specific for the p72 eukaryotic protein of African swine fever virus(ASFV)by hybridoma technique,the recombinant plasmid pCMV-p72 and B602L plasmid were co-transfected into HEK293F cells via transient transfer method for the expression and further purification of p72-His recombinant protein.Then,the purity was analyzed by SDS-PAGE and indirect ELISA.The BALB/c mice were immunized with the purified recombinant p72-His protein and anti-p72 MAbs were prepared by cell fusion and screening.After rounds of selection,four hybridoma cell lines(2B2-1,34C10,22D3 and 32H5)were found to secret MAbs against ASFV p72 protein.The isotypes of these MAbs were identified as IgG1/κor IgG2b/κ.The ascitic titers of these MAbs were higher than 6.4×104.The results of immunofluorescence showed that these 4 MAbs specifically recognized ASFV p72 protein.Further testing found that the affinity of these MAbs was 22D3>32H5>2B2-1 and34C10.Epitope competition test also revealed that these MAbs recognized different antigen binding sites.Additionally,2B2-1 and 32H5 were a good antibody pair for detection of ASFV while 34c10 was a competitor.In summary,these four hybridoma cell lines stably secreted MAbs against p72 protein with high specificity and affinity,which was beneficial to further research and development of ASFV serological diagnosis methods.

关 键 词：非洲猪瘟病毒 p72蛋白 真核表达 单克隆抗体 

分 类 号：S852.65[农业科学—基础兽医学]