Analysis The Fuction Of PTD-Foxp3 Fusion Proteins In Vitro

Foxp3,a member of the forkhead-winged-helix family of transcriptional regulators,which has been touted as a lineage-specific marker of CD4~+CD25~+ regulatory T cells(Tregs).It has been shown to be critical for the development and function of Tregs.Recently some research work had demonstrated that ectopic expression of Foxp3 could convert effector T cells into Treg-like cells such as phenotypically and functionally.The protein transduction domain(PTD),a short basic peptide fragment of transactivator(TAT) protein encoded by the human immunodeficiency virus type 1,has been developed to allow the delivery of exogenous proteins into almost all living eukaryotic cells rapidly and efficiently.The PTD fusion proteins can be located not only in cytoplasm but also in nucleus for the PTD contains a nuclear localization signal(NLS).PTD doesn't affect the bioactivities of fusion proteins.Objective:In order to acquire a lot of immnosuppression cells,biomacromolecules vehicle tool protein transduction domain(PTD) was used to deliver Foxp3 protein into cells.It may overcome the limitations of the numbers of Treg in basic research and clinical application such as autoimmune disease therapy and prevent the rejection of organ transplantation.Methods:The transduction ability and efficiency to EL-4 cells of the PTD conjugated fusion protein was analyzed by FCM and the locating sites of PTD conjugated fusion protein was detected by laser scan confocal microscope and Western Blot.The bioactivities of inhibiting cells proliferation of the fusion protein transduced CD4~+CD25~- T cells from OVA specific TCR transgenic mouse DO11.10 were analyzed by Cell Counting Kit-8(CCK-8).The mRNA levels of IL-2,INF-γ,IL-10,TGF-βand CTLA-4 were analysed by realtime RT-PCR;ELISA was used to detecte the cytokine levels of IL-2,INF-γ,IL-10 and TGF-βin the cell culture supernatant.Results:FCM assay indicated that PTD-eGFP-Foxp3 fusion protein could transduce into cells efficiently the confocal microscope indicated that PTD-eGFP-Foxp3 fusion protein not only located in cytoplasm but also in nucleus,and PTD-Foxp3 was confirmed by Western Blot.Cell proliferation assay showed that PTD-Foxp3 fusion protein could inhibit the proliferation of the CD4~+CD25~- T cells from DO11.10 mouse transduced with fusion protein.The Realtime PCR and ELISA exposed that the PTD-Foxp3 fusion protein down-regulated the levels of IL-2,IFN-γ,,but up-regulated IL-10 level compaired to Foxp3 and PTD-eGFP groups. CTLA-4 was also increased in the CD4~+CD25~- T cells from DO11.10 mouse transduced with PTD-Foxp3.Conclusion:The PTD-Foxp3 fusion protein has the ability to regulated the function of the CD4~+CD25~- T cells and convert CD4~+CD25~- T cells to Treg like cells. It may have the potential to be used as the bioregent for autoimmune therapy or prevent the transplant rejection.