Construction of three HLA-A*0201-peptide tetramers and their preliminary application in detection of vitiligo-specific cytotoxic T lymphocytes / 浙江大学学报·医学版

ABSTRACT

<p><b>OBJECTIVE</b>To construct vitiligo-specific HLA-A*0201-peptide tetramers and to apply the constructed tetramers in detection of vitiligo-specific cytotoxic T lymphocytes (CTL).</p><p><b>METHODS</b>Proteins HLA-A0201*-BSP and β2M were obtained by effective prokaryotic expression. The purified proteins were refolded with vitiligo antigen peptides MelanA 26-35, gp100 209-217, and tyrosinase 1-9, respectively to form HLA-A*0201-peptide complex. The complex was biotinylated by BirA enzyme and purified by gel-filtration chromatography. The tetramers were generated by mixing the complex with phycoerythrin (PE)-streptavidin at a ratio of 4∶1 and identified by Dot-blot assay. The capacity of tetramer to detect vitiligo-specific CTL was analyzed by flow cytometry.</p><p><b>RESULTS</b>The biotinylation of vitiligo-specific HLA-A*0201-peptide tetramers were successfully performed by Dot-blot. Flow cytometry analysis indicated that the tetramer effectively bound to specific CTL from peripheral blood of patients with vitiligo.</p><p><b>CONCLUSION</b>Three kinds of biotinylated vitiligo-specific HLA-A*0201-peptide tetramers have been constructed successfully. The tetramer can detect antigen specific CTL from patients with vitiligo.</p>