非洲猪瘟病毒p34蛋白单克隆抗体的制备及鉴定OA北大核心CSTPCD

African swine fever(ASF)is a highly contagious disease caused by African swine fever virus(ASFV)infection in domestic pigs and wild boars.ASFV encodes more than 150 proteins,of which most proteins remain to be characterized.This study aimed to express ASFV p34 protein and prepare monoclonal antibodies(mAbs)for the functional study.Firstly,the recombinant prokaryotic expression plasmid PET-21a-p34 was constructed and transformed into competent E.coli BL21(DE3)cells.The soluble His-p34 recombinant protein was expressed with IPTG induction and identified by SDS-PAGE and Western blot.The purified protein was used to immunize the mice.As a result,a hybridoma cell line capable of stably secreting the mAbs against p34 protein was obtained by cell fusion and ELISA screening.Western blot analysis showed that the mAb specifically recognized exogenous p34 protein overexpressed by transfected plasmid and endogenous p34 protein expressed by ASFV-infected porcine alveolar macrophages(PAMs).Immunofluorescence assay showed that both exogenous transfected and endogenous expressed p34 proteins were specifically labeled for visual reaction.Immunoprecipitation(IP)assay showed that the mAb specifically bound to exogenous p34 protein in HEK293T cells and endogenous p34 protein in ASFV-infected PAMs.In conclusion,the mAb to p34 protein was successfully prepared in this study,which could be used for the identification and functional study of endogenous p34 protein.