Construction of HLA-A2-peptide tetramer and application in HBV/HCV infection

• Published in: Zhong hua yi xue za zhi Vol. 84; no. 21; p. 1818
• Main Authors: Piao, Wen-hua, He, Yu, Xi, Hong-li, Sun, Xin-ting, Zhang, Heng-hui, Xu, Jing-hang, Zhao, Hong, Xu, Wen-xie, Li, Zai-liu, Wang, Gui-qiang
• Format: Journal Article
• Language: Chinese
• Published: China 02.11.2004
• Subjects: beta 2-Microglobulin - genetics; beta 2-Microglobulin - metabolism; Cloning, Molecular - methods; Female; Hepatitis B, Chronic - immunology; Hepatitis C, Chronic - immunology; HLA-A2 Antigen - genetics; HLA-A2 Antigen - immunology; Humans; Major Histocompatibility Complex - genetics; Major Histocompatibility Complex - immunology; Male; Peptide Fragments - immunology; Recombinant Fusion Proteins - genetics; T-Lymphocytes, Cytotoxic - immunology
• ISSN: 0376-2491

To construct HBV and HCV-specific HLA-A2-peptide tetramers, and to direct clinical therapy. Recombinant class I HLA-A2 heavy chains and beta-2 M were produced in Escherichia coli cells transformed with pBV220 vectors. Only the extracellular domain of class I heavy chain was expressed, following modification by replacement of the C-terminal domain with a substrate sequence for BirA biotinylation. HLA-A2-BSP was folded in the presence of beta-2 microglobulin and a specific peptide to form a peptide-MHC complex. The MHC complexes were biotinylated using purified BirA enzyme. Biotinylated MHC-peptide complexes were purified. Tetramers were generated by mixing biotinylated protein complex with streptavidin-PE at a molar ratio of 4:1. Then analysis of stained PBMCs was performed using FACScan and CellQuest software. The expression levels of pBV220-HLA-A2-BSP and beta-2M were 46% and 48% of total bacterial proteins estimated from SDS - PAGE, respectively. And they were mainly located in the insoluble fraction of the