Objective This study was aimed to develop monoclonal antibodies against CD25 protein derived from pigs, facilitating CD25 detection and exploration of the biological roles of regulatory T cells (Tregs) CD25 protein in porcine models.

Method The porcine 6×His tagged CD25 gene fragment was cloned into prokaryotic expression vector pET-28a via homologous recombination, resulting in the recombinant plasmid pET28a-CD25 construct. Then, the 6×His-CD25 recombinant protein was expressed in E. coli and the expressed recombinant protein was purified using metal chelating affinity chromatography medium (Ni-NTA) pre-packed column. The purified recombinant protein was used as antigen to immunize female BALB/c mice, and the spleen cells of immunized mice were hybridized with myeloma cells (SP2/0). The hybridized cells were screened by indirect ELISA and cloned by culturing. Finally, a large amount of monoclonal antibodies against CD25 derived from pigs were produced by mouse ascites method.

Result A single-cell-derived hybridoma cell line was established, exhibiting stable expression of CD25 monoclonal antibody. Assessment via indirect ELISA and Western blotting revealed that the CD25 monoclonal antibody exhibited exclusive reactivity towards its target protein with no cross-reactivity with other proteins, even at a high titer of 1∶4098000, thus demonstrating a strong specificity. Furthermore, indirect immunofluorescence and Western blotting assays demonstrated the monoclonal antibody could detect CD25 protein expressed by eukaryotic cells as well as endogenous CD25 protein in host spleen and lung tissues.

Conclusion The recombinant porcine CD25 recombinant protein was successfully prepared and the specific monoclonal antibody was screened, which laid a foundation for the qualitative or quantitative detection of CD25 protein in porcine Treg cellsand the study of the function of porcine Treg cells.