可溶性HLA-A*2402-pBRLF1复合物的体外折叠与四聚体化

In vitro Refolding and Tetramerization of Soluble HLA-A*2402-pBRLF1 Complex

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。

Objective To refold and construct HLA-A ＊ 2402-pBRLF1 tetramer in vitro. Methods The BirA substrate peptide （BSP） containing H chain of HLA-A s 2402 and β2 m 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 tettamer 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.