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Abstract：
We developed a highly sensitive assay for detecting protein-protein interaction using chimeric receptors comprising two molecules of interest in the extracellular domain and interferon alpha and beta receptor subunit 1 or 2 (IFNAR1/2) in the intracellular domain. This intracellular IFNAR1/2 reconstitution system (IFNARRS) proved markedly more sensitive than the NanoBiT system, currently considered one of the best detection systems for protein interaction. Employing chimeric receptors with extracellular domains from the IFNγ or IL-2 receptor and the intracellular domains of IFNAR1/2, the IFNARRS system effectively identifies low IFNγ or IL-2 levels. Cells stably expressing these chimeric receptors responded to IFNγ secreted by activated T cells following various stimuli, including a specific peptide-antigen. The activation signals were further enhanced by the expression of relevant genes, such as costimulators, via IFN-stimulated response elements in the promoters. Besides IFNγ or IL-2, the IFNARRS system demonstrated the capability to detect other cytokines by using the corresponding extracellular domains from these target cytokine receptors.
摘要：
我们开发了一种用于检测蛋白质-蛋白质相互作用的高灵敏度测定法，该测定法使用嵌合受体，该嵌合受体包含胞外结构域中的两个感兴趣的分子和胞内结构域中的干扰素α和β受体亚基1或2（IFNAR1/2）。这种细胞内IFNAR1/2重建系统（IFNARRS）明显比NanoBiT系统更敏感，目前被认为是蛋白质相互作用的最佳检测系统之一。采用具有来自IFNγ或IL-2受体的胞外结构域和IFNAR1/2的胞内结构域的嵌合受体,IFNARRS系统有效地识别低IFNγ或IL-2水平。稳定表达这些嵌合受体的细胞在各种刺激后对活化T细胞分泌的IFNγ作出反应,包括特定的肽抗原。相关基因的表达进一步增强了激活信号，例如共刺激器，通过启动子中的IFN刺激的应答元件。除了IFNγ或IL-2之外,IFNARRS系统证明了通过使用来自这些靶细胞因子受体的相应胞外结构域来检测其他细胞因子的能力。
