H-2K^b-OVA复合物的构建及体内特异性CTL的诱导

Reconstruction of H-2K^b-OVA Complex and the Specific in vivo Induction of CTL Responses

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 。

After amplification of H 2K b gene by PCR, a 15 amino acid substrate peptide for BirA dependent biotinylation was added to the COOH teminans of heavy chain. The heavy chain was over expressed in Escherchia coli as insoluble proteins after site directed mutagenesis of the H 2K b gene. Inclusion bodies were dissolved in urea,and the refolding was performed by diluting the subunits(heavy chain and β 2m)in a large volume of refolding buffer in the presence of antigenic peptide OVA 257 264 .This protein complex was used as an immunogen to induce an efficient CTL reponse in vivo.Specific CTL responses were demonstrated by H 2K b OVA tetramer staining and cytotoxicity assay. Extracellular IFN γ was also quantitatively analyzed. The results shows that specific CTL responses could be induced in vivo by H 2K b OVA complex.Generation of the tetrameric peptide MHC complexes in vitro is a powerful tool to stain specific CTL.