Constructed, Expressed Of The Ptd-hfoxp3-△prd/△fkh And Analysised Their Biological Function

The protein transduction domain(PTD),a short peptide fragment of transactivator(TAT) protein encoded by the human immunodeficiency virus type 1,has been recognized for its capacity to deliver exogenous proteins into almost all living eukaryotic cells rapidly and efficiently.The PTD fusion proteins are found to be located not only in cytoplasm but also in nucleus for the PTD containing a nuclear localization signal (NLS).It has been shown that PTD does not affect the bioactivities of fusion proteins.FOXP3,a member of the forkhead-winged-helix family of transcriptional regulators,has been identified as a lineage-specific marker for CD4+CD25+ regulatory T cells(Tregs),which is also critical for the development and function of Tregs.Recent studies have demonstrated that ectopic expression of FOXP3 can convert effector T cells into Treg-like cells both phenotypically and functionally.Thus,the functional domains in FKH domain of C terminus(ΔPRD) and rich proline of N terminus(ΔFKH) may have the repressor activity.Objective:To clone humanΔPRD andΔFKH of hFOXP3 genes fragments;To express and purify PTD-ΔPRD/PTD-eGFP-ΔPRD and PTD-ΔFKH/PTD-eGFP-ΔFKH fusion proteins and analyze their bioactivity.Methods:(1) HumanΔPRD andΔFKH of hFOXP3 genes fragments by PCR with specific primers were cloned and then inserted into pMD 18-T vector and finally verified by single and double restriction enzymes as well as sequencing analysis.(2)ΔPRD andΔFKHof hFOXP3 genes were inserted into pET28a-PTD/pET28a-PTD-eGFP vector and expressed in E.coli Rosetta(DE3) to generate fusion proteins with His tag;The fusion proteins were purified with Profinity IMAC Ni-Charged Resin,desalted by PD-10 desalting column and identified by Western blot analysis.(3) The transduction efficiency of the fusion proteins was analyzed by flow cytometry and the cellular locations of fusion proteins were detected by laser scan confocal microscopy and Western blot analysis.The bioactivities of fusion proteins in T cell proliferation were analyzed by Cell Counting Kit-8(CCK-8).(4) Levels of mRNA of IL-2 and IL-10 in PBMCs upon treatment with fusion proteins were detected by RT-PCR analysis.Results:(1) Prokaryotic expression vectors of pET28a-PTD-ΔPRD, pET28a-PTD-eGFP-ΔPRD and pET28a-PTD-ΔFKH, pET28a-PTD-eGFP-ΔFKH were correctly constructed.The fusion proteins were expressed and purified efficiently.(2) Flow cytometric analysis showed that fusion proteins could transduce into Jurkat T cells efficiently and the intracellular concentrations of fusion proteins reached a peak after PTD-eGFP-ΔFKH and PTD-eGFP-ΔPRD were co-cultured with cells for 2h at 640nm and for 4h at 1280nm,respectively.(3) Laser scan confocal microscopic studies revealed that the fusion proteins were not only located in the cytoplasm but also in the nucleus, which was further confirmed by Western-blot analysis.(4) As revealed by cell proliferation assay,PTD-ΔPRD fusion protein possessed the bioactivity to inhibit the proliferation of PBMC,indicating that FKH play an important role in hFOXP3-mediated inhibition; Furthermore,we also verified a functional domain within the PRD,which was required for hFOXP3-mediated repression.(5) Treatment with PTD-ΔPRD/ΔFKH fusion protein affected the levels of IL-2 and IL-10 mRNA expression in PBMC.In the presence of PTD-ΔPRD,and PTD-ΔFKH,PBMC showed reduced IL-2 secretion,but increased IL-10 expression. Conclusions:In summary,my studies have shown that PTD-ΔPRD and PTD-ΔFKH fusion proteins could be transduced into Jurkat E6.1T cell effectively;The results demonstrate that the PTD-ΔPRD fusion protein is a powerful reagent for inhibiting PBMC activation and proliferation; Moreover,the N-terminal of hFOXP3 is a novel functional domain;Thus, the fusion proteins may be potentially used as new therapeutic reagents for treating autoimmune disease and preventing transplantation rejection.