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.
作者
王彩霞
冯春燕
于浩洋
宋晓晖
仇松寅
刘晓飞
吴绍强
林祥梅
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)
出处
《中国动物传染病学报》
CAS
北大核心
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)。
关键词
非洲猪瘟病毒
p72蛋白
真核表达
单克隆抗体
African swine fever virus
protein p72
Eukaryotic expression
monoclonal antibody
