CD45RON-糖基化位点对galectin-3结合CD45RO突变体转染细胞的影响  

Effect of CD45RO N-glycosylation sites on galectin-3 binding to CD45RO mutant-transfected cells

作　　者：薛婧[1] 高锡强[1] 傅春燕[1] 魏强[1] 

机构地区：[1]北京协和医学院中国医学科学院医学实验动物研究所卫生部人类疾病比较医学重点实验室国家中医药管理局人类疾病动物模型三级实验室,北京100021

出　　处：《基础医学与临床》2013年第9期1165-1170,共6页Basic and Clinical Medicine

基　　金：北京协和医学院协和青年科研基金(2012J24);中央高校基本科研业务费专项资金(DWS201208)

摘　　要：目的构建CD45RO不同N-糖基化位点突变的T细胞株,并检测其与galectin-3的结合情况。方法采用N→Q定点突变技术分别去除CD45RO的11个N-糖基化位点,制备单个N-糖基化位点缺失的CD45RO,然后将其导入慢病毒表达载体pWPXL中,11个携带单个N-糖基化位点缺失的CD45RO重组慢病毒质粒与慢病毒包装质粒psPAX2和MD2.G共转染293T细胞,将包装重组慢病毒感染CD45-的J45.01细胞,流式分选后获得11株分别表达CD45RO单个N-糖基化位点缺失的J45.01 T细胞株。通过RT-PCR和流式细胞术分别从RNA水平和蛋白水平验证CD45RO突变体的转录和表达,应用流式细胞术检测CD45RO突变细胞株与galectin-3的结合情况。结果成功构建了11个CD45RO单个N-糖基化位点缺失的T细胞株,各突变细胞株能稳定表达突变基因,经测序再次证实突变位点正确,CD45RO突变体能稳定表达于细胞表面。galectin-3与N327Q突变细胞的结合明显增强,与N36Q突变细胞和N217Q突变细胞的结合明显减弱。结论 CD45RO N-糖基化位点对galectin-3与CD45RO-J45.01细胞的结合有调节作用。Objective To construct eleven cell lines which expresses CD45RO with removal of one N-glycosylation site and to examine its binding to galectin-3. Methods Individual site-directed N→Q mutagenesis of N-glycosylation site was performed for all eleven N-glycosylation sites in CD45RO and cloned into lentiviral vector pWPXL. Recombinant lentiviral was produced by 293T cells with co-transfection of pWPXL-mutated-CD45RO, the packa- ging plasmids psPAX2 and MD2. G. Human J45.01 cells were infected by the recombinant lentiviruses. The expression of CD45RO mutants was confirmed by RT-PCR and flow eytometry, and the binding of these cell lines to galectin-3 was evaluated by flow cytometry. Results Eleven cell lines each of which expressed CD45RO with removal of one N-glycosylation site were successfully constructed. RT-PCR, DNA sequencing and flow cytometry analysis revealed that the mutations were correct and CIMSRO with removal of one N-glycosylation site was stably expressed on the surface of each cell line. It was observed that galectin-3-binding to N327Q mutant was largely enhanced, to N36Q or N217Q mutant was largely suppressed. Conclusions Galectin-3 bound to CD45RO-J45.01 cells is regulated by N-glycosylation sites in CD45RO.

关 键 词：半乳糖凝集素-3 结合 N-糖基化位点 CD45RO JurkatT细胞 

分 类 号：R392.1[医药卫生—免疫学]