This study aimed to express the African swine fever virus(ASFV)capsid protein pE120R and prepare the monoclonal antibody(mAb)for the further study of its function.The pE120R gene was amplified from pCAGGS-Flag-pE120R and the prokaryotic expression plasmid pGEX-6p1-pE120R was constructed.After transforming into BL21(DE3)competent cells,it was induced to express soluble pE120R protein by IPTG.pE120R protein was purified by GST Beads and immunized BALB/c mice,the spleen cells of the immunized mice were collected and perform cell fusion with SP2/0 cell,thus a hybridoma cell line secreting pE120R monoclonal antibody was obtained by indirect ELISA.The results showed that the prokaryotic expression plasmid pGEX-6p1-pE120R was successfully constructed and the high purity pE120R protein was obtained.The results of Western blot and indirect immunofluorescence assay(IFA)showed that pE120R mAb could specifically recognize Flag-pE120R protein expressed by HEK293T cells transfected with plasmid and pE120R protein expressed by ASFV infected alveolar macrophage cells(PAMs).The binding site of this mAb is in the 30-60 aa region,and the mAb subclass identifies the heavy chain as IgG2a.In this study,the soluble pE120R protein was successfully expressed and purified;the monoantibody against pE120R was prepared,which has good specificity and laid a foundation for exploring the biological function of ASFV pE120R protein.
国家科技期刊平台
NSTL
维普
万方数据
