Cd3 Zeta Rnai Recombinant Plasmid Construction And Research On The Role Of T Cell Signal Transduction

AIM:To study the synergism effect of glycosylphosphatidyl inositol (GPI)-anchored protein CD59on CD3-mediated T cell signal transduction. METHODS:Human Jurkat cells were divided into3groups:Jurkat cells, Jurkat cells transfected with blank plasmid and Jurkat cells transfected with siRNA plasmid.CD59mRNA level was detected by RT-PCR.The cell proliferation activity of the three groups was measured by MTT colorimetry after immobilizating anti-CD3mAb and cross-linking anti-CD59mAb The phosphorylation levels of ZAP-70protein tyrosine were investigated by immunoblot analysis,and the kinetic changes of [Ca2+] in T cell endochylema were determined by Laser scanning confocal microscope imaging. RESULTS:CD59expression was successfully inhibited in transfected with siRNA plasmid group cells. The cell proliferation, phosphorylation levels of ZAP-70and degree of [Ca2+] in transfected with siRNA plasmid group cells were decreased compared with Jurkat cells and Jurkat cells transfected with blank plasmid(P<0.05),after immobilizating of anti-CD3mAb and cross-linking of anti-CD59mAb, but there was no difference between Jurkat cells and Jurkat cells transfected with blank plasmid. CONCLUSION:CD59can enhance the effect of CD3-mediated T cell signal transduction. AIM:To construct recombinant retroviral vectors CD3ζ pSUPER-RNAi that target CD3ζ gene and a stable virus-producting cell line, research the reciprocity of CD59and CD3ζ to T cell signal transduction and investigate CD59transmitting signals to T cell. METHODS:The three60nt encoded targeting CD3ζ gene shRNA sequences were cloned into pSUPER with DNA recombinant technique to construct recombinant retroviral vectors containing CD3ζ pSUPER-RNAi gene. The recombinant vectors were identified by the electrophoresis analysis of PCR, restriction enzyme digestion and DNA sequencing. The packaging cell PA317was transfected with the recombinant plasmids using Lipofectamine2000. RESULTS:The recombinant retroviral vectors containing CD3ζ pSUPER-RNAi gene was constructed correctly, which was confirmed by restriction endonuclease digestion, PCR and DNA sequencing analysis. CONCLUSION: Recombinant retroviral vectors and stable virus-producing packaging cell lines were successfully constructed and identified. It has laid a foundation for further study of expression in eukaryotic cell and the effect to T cell signal transduction. AIM:The eukaryotic expression vectors of CD3NAi and CD59RNAi were steady expressed, which is aim to research the reciprocity of CD59and CD3in T cell signal transduction and to investigate CD59transmitting signals to T cell. METHODS:The eukaryotic expression vector containing CD3NAi or CD59RNAi was identified by enzyme digestion and transfected into Jurkat cells by liposome-2000, while the steady cells cloned could get through G418screening. CD59and CD3ζ mRNA level were detected by RT-PCR. The cell proliferation activity was measured by MTT colorimetry after anti-CD3mAb and anti-CD59mAb. The phosphorylation levels of ZAP-70protein tyrosine were investigated by immunoblot analysis, and the kinetic changes of [Ca2+] in T cell endochylema were determined by Laser scanning confocal microscope imaging, while interleukin2(IL-2) in cell supernatants fluid was detected by ELISA. RESULTS: CD59and CD3Cζ expression was successfully inhibited in transfected with siRNA plasmid group cells. The cell proliferation, phosphorylation levels of ZAP-70,degree of [Ca2+] and IL-2in transfected with siRNA plasmid group cells were decreased compared with Jurkat cells after anti-CD3mAb and anti-CD59mAb (P<0.05). And the difference of siRNA plasmid group cells were statistically significant(P<0.05). CONCLUSION:The synergistic effects of CD59and CD3in singnal transduction of T cell activation, and CD59could trigger a signaling response by meanζ...