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目的: 提高可溶性HLA -A2 肽复合物体外折叠效率。方法: 在变性非还原条件下提取原核表达的HLA重链(HC)。通过离子交换及硫酸铵沉淀初步纯化后, 在β2微球蛋白(β2m)及特异性抗原肽 (Try369-377 )存在的情况下, 于pH6. 6的折叠缓冲体系中稀释复性。利用Westernblot及ELISA检测折叠产物。结果: 折叠复合物中主要含有HLA-A2 抗原肽复合物和β2m, 较少含有HC聚合体; 折叠效率较传统方法提高 2. 5倍。结论: 通过与传统方法作比较, 证实此法折叠效率比传统方法高, 为进一步HLA 肽四聚体及人工抗原提呈细胞的制备可提供足够可溶性的HLA -A2 肽单体。
Objective: To improve the folding efficiency of soluble HLA-A2 peptide complex in vitro. Methods: The prokaryotic expression of HLA heavy chain (HC) was extracted under denaturing and non-reducing conditions. After initial purification by ion exchange and ammonium sulfate precipitation, the renaturation was diluted in a pH 6.0 buffer in the presence of β2 microglobulin (β2m) and the specific antigen peptide (Try369-377). Folded products were detected by Western blot and ELISA. Results: The folded complex mainly contains HLA-A2 antigen peptide complex and β2m, containing less HC polymer; fold efficiency 2.5 times higher than the traditional method. Conclusion: Compared with the traditional methods, this method is proved to be more efficient than traditional methods in folding efficiency, which can provide sufficient HLA-A2 peptide monomer for further preparation of HLA-tetramers and artificial antigen-presenting cells.