The Research For The Fuction Of PTD-â–³NFATminiDBD Fusion Proteins In Vitro And PTD-Foxp3 Fusion Protein Inhibits Cell Proliferation And Induces Cell Apoptosis Of Mouse T Lymphoma Cell Line EL-4

Transcription factor NFAT is one of the important and key transcriptors of T cells. When the T cells were stimulated by antigen, NFAT would dephosphorylated and translocated into the nucleus. Binding with the promotor of IL-2 gene, it underwent to activate the gene transcription, then followed with the T cells activatied and the immune response. Many experiments had demostrated that IL-2 activation is dependent on the NFAT translocation.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) domain. PTD doesn't affect the bioactivities of fusion proteins.Objective: To evaluate the bioactivities and elucidated the mechanisms of functions of the fusion protein of PTD conjugated mutant of NFAT mini DNA binding domain (PTD-△NFATminiDBD) on T cells activation, proliferation and their immune functions, and to settle the foundation of application of the fusion protein for preventing and treating organ rejection and autoimmune diseases.Methods: The transduction efficiency of the fusion proteins were analyzed by FACS and the location of fusion proteins were detected by laser scan Confocal microscope and evaluated by western-blot locate. The inhibition of T cell proliferation of the fusion proteins were analyzed by Cell Counting Kit-8 (CCK-8). The expression levels of mRNA of IL-2 in activiated T cells were detected by RT-PCR. IL-2 reporter gene assay and Enzyme-linked immunosorbent assay were used to detecte the IL-2 expression levels in activiated T cells.Results: FACS assay indicated that fusion proteins could transduce into Jurkat cells efficiently and the intracellular fusion proteins concentration got to the peak after the proteins were co-cultured with cells for 2h at 2.56μM. The laser scan Confocal microscope showed that the fusion proteins not only located in cytoplasm but also in nucleus. It was also confirmed by western-blot. Cell proliferation assay displayed that PTD-△NFATminiDBD fusion protein could inhibit the proliferation of T cells. The RT-PCR, ELISA and IL-2 reporter gene assay indicated that PTD-△NFATminiDBD fusion protein could obviously inhibit the amount of IL-2 not only at the transcriptional level but also the protein level in the activated T cells.Conclusion: The PTD-△NFATminiDBD fusion protein could transducet into the T cells and located in the nucleus and inhibited the T cell activated. Object: To investigate the effect of PTD-Foxp3 fusion protein on cell proliferation and cell apoptosis of mouse T lymphoma cell line EL-4.Methods: The cell proliferation was detected by Cell Counting Kit-8 (CCK8) and the percentage of apoptotic cells was determined by Annexin V-FITC and PI double staining and flow cytometry.Results: Cell proliferation assay shown that PTD-Foxp3 fusion protein could inhibit the proliferation of EL-4 cells with a dose dependent manner; Flow cytometry assay results demonstrated that PTD-Foxp3 fusion protein can induce apoptosis of EL-4 cells.Conclusion: The PTD-Foxp3 fusion protein inhibits cell proliferation of EL-4 cells significantly and induce apoptosis in EL-4.