【摘要】 目的研究可溶性HLA-A*2402-pBRLF1复合物的体外折叠与四聚体化。方法将原核高效表达的可溶性HLA-A*2402-BSP融合蛋白及β2微球蛋白(β2m)与HLA-A*2402限制性抗原肽Epstein-Barr病毒(EBV)即刻早期BRLF1蛋白中的九肽NH2-DYNFVKQLF-COOH进行稀释复性折叠,形成HLA-A*2402-pBRLF1复合物单体,并在BirA酶的作用下进行生物素化,使生物素结合到HLA-A*2402-pBRLF1复合物中H链C端的BSP序列上形成生物素化的可溶性HLA-A*2402-pBRLF1复合物单体。利用特异单抗(W6/32和兔抗人β2m抗体)及链霉亲合素进行ELISA和Western blot,检测稀释复性和生物素化的折叠产物。然后用此生物素化的单体分子进一步构建成HLA-A*2402-pBRLF1四聚体来检测人工抗原提呈细胞(aAPCs)诱导的特异性细胞毒T淋巴细胞(CTL)。结果折叠复合物中,主要含有HLA-A*2402-pBRLF1复合物单体及β2m两种成分,其中HLA-A*2402-pBRLF1复合物单体可生物素化和四聚体化。应用HLA-A*2402-pBRLF1四聚体对特异性CTL检测结果说明体外成功地构建了HLA-A*2402-pBRLF1四聚体。结论HLA-A*2402-pBRLF1四聚体构建成功,为特异性CTL的检测提供了有力的工具。更多还原

【Abstract】 Objective To refold and construct HLA-A*2402-pBRLF1 tetramer in vitro.Methods The BirA substrate peptide（BSP） containing H chain of HLA-A*2402 and β₂m was expressed highly as insoluble aggregates in E.coli,and then the two subunits were refolded to form an HLA-A*2402-pBRLF1 complex by dilution method in the presence of an antigenic peptide（NH2-DYNFVKQLF-COOH of EB virus BRLF1）.The BirA enzyme was used to biotinylate the refolded complex.The refolded and biotinylated products were detected by ELISA and Western blot with monoclonal antibody(W6/32 and rabbit anti-human β₂m antibody) and streptavidin.The complex was used to construct HLA-A*2402-pBRLF1 tetramer by which specific CTL induced by artificial antigen presenting cells（aAPCs） were demonstrated.Results HLA-A*2402-pBRLF1 tetramer could be constructed successfully in vitro.Conclusion The refolding and tetramerization of HLA-A*2402-pBRLF1 complex were successfully acquired and proved by our practical immunological methods.Generation of the HLA-A*2402-pBRLF1 tetramer is a powerful tool to stain specific CTL.更多还原