Preparation of Monoclonal Antibody against Porcine CD56 and Preliminary Identification of Its Epitope
As an important surface molecule of NK cells,CD56 plays an important role in the de-velopment,subgroup typing and function of NK cells.Therefore,in order to facilitate the screening of different subpopulations of porcine NK cells and explore their biological functions,this study prepared porcine CD56 monoclonal antibodies and preliminically identified their epitopes.Firstly,the amino acid sequences of the dominant epitope of porcine CD56 protein ex-tracellular region(50-499aa)was predicted and selected by bioinformatics software.Then the se-lected region of CD56 was expressed by prokaryotic expression system,and the protein was puri-fied and renaturated.After that,the recombinant protein was immunized to BALB/c mice,and a hybridoma cell line with stable secretion of porcine CD56 monoclonal antibody was obtained through cell fusion and subcloning experiments,which was named 1G8.Western blot results showed that the prepared porcine CD56 monoclonal antibody could bind specifically to the total protein of porcine brain and spleen.To further explore the specificity of CD56 monoclonal anti-body,NK cells were isolated from porcine peripheral blood,then indirect immunofluorescence assay(IFA)was performed by using the prepared CD56 monoclonal antibody as the primary anti-body.The results showed that the prepared monoclonal antibody could bind specifically to NK cell membrane.Finally,in order to further investigate the epitopes recognized by monoclonal an-tibodies,we further truncated the selected porcine CD56 gene into four truncations(50aa-192aa,193aa-294aa,295aa-397aa,398aa-499aa),which were insert into eukaryotic expression plasmids.And then these eukaryotic expression recombinant plasmids were transfected into 293T cells,re-spectively.Western blotting and IFA results showed that the epitopes recognized by the prepared CD56 monoclonal antibody were 193aa-294aa of porcine CD56 protein.In this study,porcine CD56 monoclonal antibody was successfully prepared,and its specificity and epitopes were preli-minically identified,which is of great significance for screening porcine NK cell subsets and ex-ploring the biological function of porcine NK cells
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