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本研究的目的是为得到表达人MHCⅠ类分子轻链蛋白β2m、β2m的C-末端与一段五聚体(penta)序列相连、且共价结合一段荧光素编码序列(GFP)的五聚重组人β2m-GFP蛋白。采用PCR扩增GFP基因及β2m基因片段,将其克隆到原核表达载体,并通过IPTG诱导目的基因在大肠杆菌中表达融合蛋白。提纯重组蛋白,并用SDS-PAGE和蛋白印迹法鉴定。结果表明,成功将GFP基因或人β2m基因片段插入含五聚结构域的PVT2载体。转化菌经诱导后,SDS-PAGE显示有新生的蛋白表达条带,分子量与预期的大小基本一致。重组蛋白用Ni-NTA柱提纯,纯化蛋白的蛋白质经SDS-PAGE分析可见单一条带。Western blot显示重组蛋白具有良好的免疫活性。因此,我们获得了含五聚结构域的人β2m-GFP在原核系统中的稳定表达,为后续深入研究含五聚结构域的重组MHCⅠ类分子的功能及应用奠定了基础。
The purpose of this study was to obtain a pentameric recombinant human co-expressing a segment of the fluorescein-encoding sequence (GFP) that is linked to a penta sequence at the C-terminus of the human MHC class I molecule light chain beta 2m, β2m-GFP protein. The GFP and β2m gene fragments were amplified by PCR, cloned into the prokaryotic expression vector and induced by IPTG to express the fusion protein in E. coli. Recombinant proteins were purified and identified by SDS-PAGE and Western blotting. The results showed that the GFP gene or human β2m gene fragment was successfully inserted into the pentamer domain-containing PVT2 vector. After induction of transformed bacteria, SDS-PAGE showed a new protein expression band, the molecular weight and the expected size is basically the same. The recombinant protein was purified by Ni-NTA column. The purified protein showed single band by SDS-PAGE. Western blot showed that recombinant protein has good immunocompetence. Therefore, we obtained the stable expression of human β2m-GFP containing pentameric domain in prokaryotic system, which lays the foundation for the further study of the function and application of pentameric domain-containing recombinant MHC class I molecules.