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在扩增了H 2Kb 基因后 ,将可生物素化的序列连接到其胞外区末端 ,构建成可溶性重组分子 ,进行原核表达。通过定点突变获得了高表达的融合蛋白 ,再利用 6×Histidinetag进行蛋白纯化 ,在对H 2Kb分子高亲和力的OVA2 57 2 64肽及 β2 m蛋白存在下 ,三者体外折叠成具有完整构象的H 2Kb OVA类分子复合物。用其作为免疫原诱导特异性CTL ,并构建H 2Kb OVA四聚体检测特异性CTL。应用H 2Kb OVA四聚体对特异性CTL检测结果与经典的细胞毒试验一致 ,同时对胞外IFN γ进行了检测。说明H 2Kb OVA复合物可有效诱导体内特异性CTL反应 ,在体外构建MHC I类分子四聚体 ,为特异性T细胞的检测提供了有利的工具
After amplifying the H 2Kb gene, the biotinylated sequence was ligated to the end of its extracellular region to construct a soluble recombinant molecule for prokaryotic expression. Highly expressed fusion protein was obtained by site-directed mutagenesis and then was purified by 6 × Histidinetag. In the presence of OVA2 57 2 64 peptide and β2 m protein with high affinity for H 2Kb, the three proteins were folded in vitro into H 2Kb OVA Molecular Complex. Use it as an immunogen to induce specific CTLs and construct H 2Kb OVA tetramers to detect specific CTLs. The results of CTL detection using H 2Kb OVA tetramer were in accordance with the classical cytotoxicity assay, and the detection of extracellular IFN γ was also carried out. These results suggest that the H 2Kb OVA complex can effectively induce specific CTL responses in vivo and construct MHC class I tetramer in vitro, which provides an advantageous tool for the detection of specific T cells