【摘要】 目的:研究FTY720诱导1型1-磷酸鞘氨醇受体(S1P1)内化与后者保守基序之间的关系。方法:以HA-S1P1(WT)-Myc-EGFP-N1融合表达载体为模板,应用重叠PCR的方法,将S1P1保守基序ERY突变为ENY,构建HA-S1P1(R142N)-Myc-EGFP-N1融合表达载体。测序鉴定后,经Polyfect转染入HEK293细胞。G418筛选出稳定细胞株。100 nmol/LFTY720处理3、6、12小时后,荧光显微镜下观察S1P1在HEK293细胞上的表达情况。结果:HA-S1P1(R142N)-Myc-EGFP-N1载体被成功构建,S1P1(WT)和S1P1(R142N)蛋白表达在HEK293稳定细胞株的表面,FTY720能诱导S1P1(WT)内化,但不能诱导S1P1(R142N)内化。结论:FTY720诱导S1P1内化与ERY保守基序有关。更多还原

【Abstract】 Objective:To study the relation between conserved motif of sphingosine 1-phosphate receptor type 1(S1P1) and FTY720-induced internalization.Methods:With HA-S1P1(WT)-Myc-EGFP-N1 fusion vector as template,HA-S1P1(R142N)-Myc-EGFP-N1 fusion vector was constructed by overlap PCR.The conserved ERY motif of wild type S1P1 was mutated into ENY.The recombinant vectors were confirmed by sequencing,then they were transfected into HEK293 cells by Polyfect.The transfected HEK293 cells were selected with G418.Cells were incubated for 3,6,12 hours in the absence or presence of 100 nmol/L FTY720,then S1P1 gene expression was analyzed by fluorescence microscopy.Results:Sequencing confirmed HA-S1P1(R142N)-Myc-EGFP-N1 vectors was successfully constructed.S1P1(WT) protein and S1P1(R142N) protein were expressed on the stably transfected-HEK293 cell surface.FTY720 induced S1P1(WT) internalization,but not S1P1(R142N).Conclusion:FTY720-induced S1P1 internalization is related with conserved ERY motif.更多还原