Abstract:
Objective The monoclonal antibody against CD25 in pigs was obtained to facilitate detecting and studying the regulatory T cells (Tregs).
Method The porcine 6×His tagged CD25 fragment was cloned into prokaryotic expression vector pET-28a via homologous recombination to construct the recombinant plasmid pET28a-CD25. Subsequently, the 6×His-CD25 recombinant protein was expressed in E. coli and purified on a metal chelating affinity chromatography medium (Ni-NTA) pre-packed column to be used as the antigen to immunize female BALB/c mice. Spleen cells of the immunized mice were hybridized with myeloma cells SP2/0 and screened by indirect ELISA prior to culture cloning. The target monoclonal antibody against CD25 was thus prepared using mouse ascites method.
Result A single cell-derived hybridoma cell line with a stable expression of CD25 monoclonal antibody was successful obtained. ELISA and western blotting showed the antibody had a strong specificity with an exclusive reactivity towards the target protein, not the others, even at a high titer of 1:4098000. In the indirect immunofluorescence and western blotting assays, the antibody not only detected CD25 in the eukaryotic cells but also endogenous one in the host spleen and lung tissues.
Conclusion The recombinant porcine CD25 was successfully prepared and the specific monoclonal antibody obtained for qualitative and/or quantitative detection of the protein as well as further studies on the functions of porcine Tregs.