Study Of The Synergistic Effects Of CD59,CD46in Singnal Transduction Of T Cell

Objectives:To study the synergistic effect of glycosylphosphatidyl inositol (GPI)-anchored protein CD59on CD46-mediated T cell signal transduction.Methods: Two pairs of single-stranded oligo encoding CD59shRNA sequence were synthesized and inserted into lined pSUPER expression vectors to construct recombinant vectors. Then E. coli JM109was transfected with two recombinant vectors and one negative-control vector (siCD59-C).Human Jurkat cells were divided into4groups: Jurkat cells(I group)、Jurkat cells transfected with pSUPER plasmid(Ⅱ group)、 Jurkat cells transfected with pSUPER-CD59siRNA plasmid(III group) and Jurkat cells transfected with pSUPER-CD46siRNA plasmid(IV group) by Lipofectamne2000.Stable expression clones were selected by the addition of G418.CD59and CD46mRNA levels was detected by RT-PCR. After crosslinking of CD46and CD59antibodies,T cell proliferation was measured by MTT assay (lymphocyte transformation test);the phosphorylation levels of ZAP-70were investigated by Western Blot.Results:The results of PCR, enzyme digestion and sequence demonstrated that the target sequence had been successfully inserted into the vector. RT-PCR demonstrated that mRNA expression of CD59in II group and CD46in III group were significantly inhibited Compared with I、Ⅱ group (P<0.05).After crosslinking anti-CD46mAb and anti-CD59mAb, the proliferation of lymphocyte and ZAP-70protein phosphorylation bands gray value in I group were evidently increased compared with Ⅱ、 Ⅲ group (P<0.05), but that in II group was lower than III group (P<0.05). There was no difference between I and II group (P>0.05).Conclusions:Recombinant vector pSUPER-siCD46and pSUPER-siCD59respectively inhibited the expression of CD46and CD59.Experiment demonstrated that co-expression of two proteins(CD59,CD46) are more effective in cell proliferation、ZAP-70phosphorylation. Hence, CD59and CD46have synergistic effects in signal transduction of T cell activation, which may provide a very promising strategy to study the mechanism of GPI(glycosylphosphatidyl inositol)anchored protein mediated T cell signal transduction. The research opens up a new way for gene targeting treatment of T cell leukemia that has important theoretical significance and clinical application prospects.