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目的用前期构建好的T细胞活化连接蛋白(LAT)-EGFP质粒,基因点突变技术使LAT-EGFP的棕榈酰化位点突变,使LAT-EGFP-M(棕榈酰化位点突变的LAT-EGFP)的细胞膜定位功能缺失,抗体交联CD59后观察T细胞信号活化状态的改变。方法采用电转染的方法转染Jurkat细胞,抗体交联CD59后,共聚焦荧光显微镜下观察LAT分布情况的变化;用噻唑蓝(MTT)比色法检测抗体交联前后各组Jurkat细胞增殖情况差别;应用ELISA检测各组细胞上清中IL-2的变化水平;用Western blot技术检测抗体交联后LAT-EGFP和LAT-EGFP-M的磷酸化程度。结果抗体交联CD59之后,LAT-EGFP-M较LAT-EGFP相比不能定位聚集于CD59所在的脂筏区域,并且转染了突变LAT-EGFP-M质粒的Jurkat细胞增殖速度和IL-2分泌能力低于转染LAT-EGFP质粒、转染空载体EGFP-N3及未转染的细胞。LAT-EGFP-M的磷酸化程度远低于LAT-EGFP。结论棕榈酰化位点缺失的LAT会减弱CD59在T细胞中的信号转导功能。
OBJECTIVE: To construct the LAT-EGFP plasmid by using the pre-stage LAT-EGFP plasmid. Mutagenesis of the LAT-EGFP palmitoylation site by gene point mutagenesis resulted in the formation of LAT-EGFP-M (palmitoylation site-mutated LAT- EGFP) cell membrane locating function is missing, antibody cross-linked CD59 observed T cell activation signal changes. Methods The Jurkat cells were transfected by electrotransfection and the cross-linking of CD59 was observed by confocal laser scanning microscope. The proliferation of Jurkat cells was detected by MTT assay The level of IL-2 in the supernatant of each group was detected by ELISA. The phosphorylation of LAT-EGFP and LAT-EGFP-M was detected by Western blot. Results After antibody cross-linking of CD59, LAT-EGFP-M was unable to locate and accumulate in the lipid raft region where CD59 is located as compared with LAT-EGFP, and Jurkat cells transfected with the mutant LAT-EGFP-M plasmid proliferate and IL-2 secretion The ability of transfecting LAT-EGFP plasmid was lower than that of transfecting empty vector EGFP-N3 and untransfected cells. The phosphorylation of LAT-EGFP-M is much lower than that of LAT-EGFP. Conclusion LAT with a lack of palmitoylation sites attenuates the signal transduction of CD59 in T cells.