可溶性单链MHC-肽四聚体的构建研究

Preparing of the single-chain MHC-peptide complex tetramer

目的:构建检测抗原特异性细胞毒性T淋巴细胞的单链MHC-肽四聚体。方法:通过在MHC重链基因中引入生物素化位点,并在轻链和重链基因中引入同一限制性酶切位点连接MHC轻链和重链构建可生物素化的单链MHC分子。结果:重组子以包涵体形式表达得到单链MHC分子,重折叠、生物素化、荧光标记后得到活性单链MHC-肽四聚体。结论:单链MHC分子操作简便,提高了四聚体构建效率。

Objective：To construct a single chain MHC-epitope tetramers for detecting epitope specific cytotoxic T lymphocyte. Motheds：The C terminus of β2m fusing with extracellular domains of HLA A0201 was connected with a accessional BirA substrate peptide by a linker through restriction enzyme digestion and hgation. Results：Expression of the recombinant in a pET systerm as inclusion body was followed by refolded in the presence of the antigenic peptide. After biotinylization of the MHC-peptide complex, the tetramers was obtained by the coupling of a fluorchrome conjugated streptavidin with the complex. Conclusion：The novel method of constitution of tetramers was more efficient than the old labor-consuming one.