| Objective:Constructing the fusion expression vector of CD59 gene and CD2 gene, which was steady expressed, is aim to research the reciprocity of CD59 and CD2 in T cell signal transduction and investigate CD59 transmitting signals to T cell.Methods:CD59 and CD2 genes were cloned by RT-PCR from the total RNA of Jurkat cell. CD59-T and CD2-T cloning vector were respectively constructed and cut by restriction endonuclease. They were cloned into the vector plRES to construct a eukaryotic expression vector containing CD59-linker-CD2 fusion gene. The vector was transfected into Jurkat cells by liposome, while steady cells cloned could get through G418 screening. CD59 gene was detected by immunoenzyme method, RT-PCR and Western Blot. After CD59mAb cross-linking, the signal molecule of phosphorylated ZAP70 was tested by Western Blot, while interleukin 2 (IL-2) in cell supernatants fluid was detected by ELISA.Results:The eukaryotic expression vector containing CD59-linker-CD2 fusion gene was constructed correctly, which was confirmed by restriction endonuclease digestion, PCR and DNA sequencing analysis. The results of immunoenzyme method, RT-PCR and Western Blot indicated that CD59 gene was highly expressed in transfected Jurkat cells compared with normal Jurkat cells, while the differences were statistically significant(P<0.05). ZAP70 and IL-2 were highly expressed in transfected Jurkat cells compared with normal Jurkat cells, the differences were statistically significant(P<0.05).Conclusions:The eukaryotic expression vector containing CD59-linker-CD2 fusion gene could steady expressed in Jurkat cells. After CD59mAb cross-linking, CD59 could trigger a signaling response by means of CD2, which caused a series of changes of cell signal molecules. Our results lay a foundation for researching the synergistic effect of CD59 and CD2 in T cell signal transduction and CD59 transmitting signals to T cell. |
