[可溶性 HLA - A2 - 肽复合物的体外重折叠与生物素化]

[In-vitro refolding and biotinylation of soluble HLA-A2-peptide complex].

作者信息

Wong Xiu-fang, Chen Hao, Wu Xiong-wen, Liang Zhi-hui, Han Jun-yan, Huan Ya-fei, Gong Fei-li

机构信息

Department of Immunology, Tongji Medical College, Huazhong Sciences and Technology University, Wuhan 430030, China.

出版信息

Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2004 May;20(3):265-8.

PMID:15193213

Abstract

AIM

To refold and biotinylate HLA-A2-peptide complex in-vitro.

METHODS

The BirA substrate peptide (BSP) containing H chain of HLA-A2 and beta(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 beta(2m) antibody and streptavidin.

RESULTS

The refolded complex was composed of H chain aggregate, HLA-A2-peptide complex and beta(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.

摘要

目的

在体外对HLA - A2 - 肽复合物进行重折叠和生物素化。

方法

含HLA - A2重链和β2微球蛋白的BirA底物肽（BSP）在大肠杆菌中以不溶性聚集体形式高效表达，然后在抗原肽（EB病毒潜伏膜蛋白2A LMP2A的NH2 - CLGGLLTMV - COOH）存在下，通过稀释法将两个亚基重折叠形成HLA - A2 - 肽复合物。随后用BirA酶对重折叠的复合物进行生物素化。用单克隆抗体W6/32、兔抗人β2微球蛋白抗体和链霉亲和素通过ELISA和Western印迹法检测重折叠和生物素化的产物。