Treg细胞中关键转录因子Foxp3相互作用蛋白的鉴定与分析

Identification of the Foxp3-interacting proteins in mice Treg cells

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。

This study was designed to identify the components of Foxp3 protein complex. Firstly, we prepared the tags-knock-in mice with streptavidin binding peptide（SBP） and protein G binding domain fused before the stop codon of exon 14 in Foxp3 gene. The Treg cells were sorted by fluorescence activated cell sorting（FACS） and the Foxp3 interacting protein complex was isolated by an affinity magnetic beads purification（TAP） strategy. The components of the complex were then analyzed by mass spectrum（MS）, and the interactions between the selected candidate components were analyzed by co-immunoprecipitation. We found that the purity of the isolated Treg cells was above 90%. The MS assay revealed over 100 candidates interacting proteins of Foxp3 protein. And several candidates Foxp3 complex components could interact with each other. Therefore, in this study we reveal the candidate Foxp3 interacting proteins, which however, have to be determined for their interaction each other in the future.