Effect of PTD-ΔPRD Foxp3 fusion protein on the mixed lymphocyte reaction in mice / 中南大学学报(医学版)

ABSTRACT

Objective To express protein transduction domain (PTD)-deletion proline domain (ΔPRD) Foxp3 fusion protein, and to analyze its influence on mixed lymphocyte reaction in mice.Methods We cloned mouse ΔPRD of Foxp3 gene by PCR, and inserted it into pET28a-PTD, pET28a-PTD-eGFP vector, then expressed fusion proteins in E.coli Rosetta (DE3). The fusion proteins were purified and refolded by Profinity IMAC Ni~(2+)-Charged Resin. The expression of fusion proteins was identified by Western blot. Flow cytometry assay was used to detect the effect of PTD-ΔPRD fusion protein to transduce into mouse EL-4 cells. The ability of fusion protein to inhibit the proliferation of EL-4 cells was analyzed by two-way mixed lymphocyte reaction.Results The PTD-ΔPRD fusion proteins were expressed and purified efficiently. Western blot and flow cytometry indicated that PTD-ΔPRD fusion protein was transduced into EL-4 efficiently. Mixed lympocyte reaction assay showed that PTD-ΔPRD fusion protein had the bioactivity to inhibit the proliferation of EL-4 cells.Conclusion The PTD-ΔPRD fusion protein was expressed in E.coli system and could be transduced into cells effectively, suggesting that PTD-ΔPRD fusion protein may be an inhibitor in lymphocytes from mouse spleen.