Prokaryotic Expression and Monoclonal Antibody Preparation of Bovine Promyelocytic Leukemia Protein
[Objective]Bovine promyelocytic leukemia protein(PML),a constituent of nuclear substructure called PML nuclear bodies(PML NBs),was crucial for antiviral intrinsic immunity.In this study,monoclonal antibody against bovine PML was generated to facilitate further investigation into the structure and function of PML NBs.[Method]Recombinant plasmid pET-32a-bPML expressing bPML gene truncator was constructed.The recombinant plasmid was transformed into Escherichia coli Transetta(DE3)competent cells,and induced by IPTG,and the expression products were analyzed by SDS-PAGE and Western blotting.The recombinant protein purified by nickel column affinity chromatography was utilized for immunization of mice,and monoclonal antibody was prepared.ELISA was applied to determine the classes/subclasses and subtypes of the monoclonal antibody,and the epitope recognized by the monoclonal antibody was further identified.The specificity of monoclonal antibody was detected by detecting cells of different species origin.The monoclonal antibody against bovine PML was analyzed by Western blotting and indirect immunofluorescence assay(IFA)to detect of exogenously transfected bovine PML cells.IFA established by this monoclonal antibody was used to detect the endogenous PML localization in different bovine-derived cells.[Result]Bovine PML was solubly expressed in Escherichia coli with a molecular weight size of 50 ku,a monoclonal antibody targeting bovine PML,bPML-2G5,was prepared from this purified protein as an immunogen.bPML-2G5 belonged to the IgG1 subclass,its light chain was Kappa type.The epitope recognized by bPML-2G5 was located in the structural domain 78 EQPRPSTSRA88 of the bovine PML.Western blotting and IFA analysis demostrated that bPML-2G5 not only specifically recognized the exogenously transfected bovine PML,but also specifically recognized PML localized within PML NBs in bovine kidney cells,bovine nasal turbinate cells,and bovine embryonic tracheal cells.[Conclusion]In this study,a hybridoma cell line secreting bovine PML monoclonal antibody was successfully prepared,and its secreted monoclonal antibody could be used to detect exogenously and endogenously expressed bovine PML proteins.
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