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目的鉴定Treg细胞中Foxp3相互作用蛋白。方法构建包含SBP和Protein G结合肽的Tags的Foxp3 knock-in转基因小鼠,分选Treg细胞,用亲和磁珠法纯化Foxp3相互作用蛋白复合体,质谱测序鉴定复合体蛋白组分并进行蛋白质组学分析和蛋白相互作用的验证。结果流式细胞分选的Treg纯度超过90%;经过亲和磁珠法纯化和质谱分析显示Foxp3有100个以上候选相互作用蛋白伙伴;免疫共沉淀方法初步鉴定部分伙伴蛋白间可相互结合。结论本研究在小鼠体内Treg细胞中初步获得Foxp3候选相互作用蛋白并证实部分蛋白间可相互作用,为这些蛋白对Treg细胞的功能鉴定提供基础。
Objective To identify Foxp3 interacting proteins in Treg cells. Methods Foxp3 knock-in transgenic mice containing Tags of SBP and Protein G binding peptides were constructed and Treg cells were sorted. Foxp3 interacting protein complexes were purified by affinity magnetic beads. Mass spectrometry sequencing identified the complex protein components and performed protein Group analysis and validation of protein interactions. Results The purity of Treg by flow cytometry was over 90%. After purification by affinity magnetic beads and mass spectrometry analysis, Foxp3 had more than 100 candidate interacting protein partners. Co-immunoprecipitation assay showed that some of the partner proteins could be combined with each other. Conclusion In this study, Treg cells in mice preliminarily obtained Foxp3 candidate interacting protein and confirmed that some of the proteins can interact with each other, providing a basis for the functional identification of Treg cells by these proteins.