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目的:探讨高迁移率蛋白1(high mobility protein box1,HMGB1)与活化T细胞核因子2(nuclearfactor of activating T cells2,NFAT2)之间的直接相互作用。方法:首先观察HMGB1与NFAT2在细胞外的相互作用,构建pET28a(+)-HMGB1、pGEX-KG-NFAT2质粒,应用网织红细胞系统进行体外翻译得到带有放射性硫同位素35S的His-HMGB1融合蛋白。应用蛋白纯化技术得到的GST-NFAT2融合蛋白,利用GST-pulldown实验检验二者在体外是否可直接结合;然后观察HMGB1与NFAT2在细胞内能否直接结合,构建HMGB1和NFAT2的真核表达质粒,共同转染人胚胎肾293T细胞系,刺激之后裂解细胞,应用相应的抗体进行免疫共沉淀检测,观察二者在细胞内直接结合的情况。结果:利用GST-pull down实验及免疫共沉淀实验证实,HMGB1全长蛋白与NFAT2全长蛋白在细胞内、外有直接结合的条带,而对照组没有结合条带,说明HMGB1可与NFAT2特异性结合。结论:HMGB1与NFAT2之间存在直接相互作用。
Objective: To investigate the direct interaction between high mobility protein box 1 (HMGB1) and nuclear factor of activating T cells 2 (NFAT2). Methods: The extracellular interaction between HMGB1 and NFAT2 was observed. Plasmids pET28a (+) - HMGB1 and pGEX-KG-NFAT2 were constructed and the His-HMGB1 fusion protein with radioactive sulfur isotope 35S was translated by in vitro reticulocyte system . The GST-NFAT2 fusion protein obtained by the protein purification technique was used to test whether GST-NFAT2 could be directly bound in vitro by GST-pulldown assay. Then, whether HMGB1 and NFAT2 could directly bind to each other in the cell and construct eukaryotic expression plasmids of HMGB1 and NFAT2, The 293T cells were co-transfected into human embryonic kidney 293T cells. After stimulated, the cells were lysed and the corresponding antibodies were co-immunoprecipitated to observe the direct binding of the two in cells. Results: The results of GST-pull down assay and coimmunoprecipitation showed that HMGB1 full-length protein and NFAT2 full-length protein had a direct binding band in the cell and outside, while the control group had no binding band, indicating that HMGB1 was specific to NFAT2 Sexual bonding. Conclusion: There is a direct interaction between HMGB1 and NFAT2.