摘要
目的 :可溶性HLA A2 抗原肽复合物的体外折叠复性与生物素化。方法 :原核高效表达的HLAH链及 β2m,在抗原肽 (EB病毒的膜潜伏蛋白LMP2A的NH2 CLGGLLTMV COOH残基 )的存在下 ,通过稀释复性折叠成HLA A2 抗原肽复合物 ,并在BirA酶的作用下进行生物素化 ,使生物素结合到HLA A2 抗原肽复合物中的H链C端。利用特异性抗体 (mAbW6 / 32和兔抗人 β2m抗体 )及链霉亲合素进行ELISA和Westernblot,检测稀释复性和生物素化的折叠产物。结果 :折叠复合物中 ,主要含有HLAH链聚合体、HLA A2 肽复合物单体及 β2m3种成分 ,其中H链聚合体与HLA A2单体可生物素化。结论 :成功地获得生物素化的HLA A2 抗原肽复合物单体 ,为进一步构建四聚体及制备人工抗原提呈细胞奠定了基础 ,也为过程复杂的HLA
AIM: To refold and biotinylate HLA A2 peptide complex in vitro . METHODS: The BirA substrate peptide (BSP) containing H chain of HLA A2 and β 2m were expressed highly as insoluble aggregates in E.coli , and then the two subunits were refolded to form an HLA A2 peptide complex by dilution method in the presence of an antigenic peptide (NH 2 CLGGLLTMV COOH of EB virus latent membrane protein 2A LMP2A). Then the BirA enzyme was used to biotinylate the refolded complex. The refolded and biotinylated products were detected by ELISA and Western blot with mAb W6/32 and rabbit anti human β 2m antibody and streptavidin. RESULTS: The refolded complexe was composed of H chain aggregate, HLA A2 peptide complex and β 2m . Both HLA A2 peptide complex and the H chain aggregate could be biotinylated. CONCLUSION: The refolding and biotinylation of HLA A2 peptide complex were successfully performed and the products were confirmed by our practical immunological method. This study laid the foundation for the preparation of HLA peptide tetramer and artificial antigen presenting cells.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2004年第3期265-268,共4页
Chinese Journal of Cellular and Molecular Immunology
基金
国家自然科学基金资助项目 (No .30 2 71 2 0 1 )
国家重点基础研究发展规划 (973)资助项目(No.2 0 0 1CB51 0 0 0 8)
