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

发布时间：2018-11-29 07:43

【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。
[Abstract]:Aim to construct T cell lines with different N-glycosylation sites of CD45RO and to detect their binding to galectin-3. Methods 11 N-glycosylation sites of CD45RO were removed by N Q site-directed mutagenesis, and a single CD45RO, with missing N-glycosylation sites was prepared and then introduced into lentivirus expression vector pWPXL. Eleven CD45RO recombinant lentivirus plasmids carrying a single N-glycosylation site deletion were co-transfected with lentivirus packaging plasmids psPAX2 and MD2.G into 293T cells. J45.01 cells infected with CD45- were infected with the recombinant lentivirus. After flow sorting, 11 J45.01 T cell lines expressing CD45RO single N-glycosylation site deletion were obtained. The transcription and expression of CD45RO mutants were verified by RT-PCR and flow cytometry from RNA level and protein level respectively. The binding of CD45RO mutant cell line to galectin-3 was detected by flow cytometry. Results A total of 11 T cell lines with single N-glycosylation site deletion in CD45RO were successfully constructed. The mutant genes were stably expressed in each mutant cell line, and the mutation sites were confirmed to be correct by sequencing. The binding of galectin-3 to N327Q mutant cells was significantly enhanced, while the binding to N36Q mutant cells and N217Q mutant cells was weakened. Conclusion CD45RO N-glycosylation sites can regulate the binding of galectin-3 to CD45RO-J45.01 cells.
【作者单位】：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;
【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)
【分类号】：R392.1

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