Zhejiang da xue xue bao. Yi xue ban = Journal of Zhejiang University. Medical sciences · 2014
[Construction of three HLA-A*0201-peptide tetramers and their preliminary application in detection of vitiligo-specific cytotoxic T lymphocytes].
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Wu Jun-qi, Shi Jia, Dong Jian-feng, Jiang Yi-xiu, Zhuang Sun-hong, Yang Jun
Аннотация
OBJECTIVE: To construct vitiligo-specific HLA-A*0201-peptide tetramers and to apply the constructed tetramers in detection of vitiligo-specific cytotoxic T lymphocytes (CTL).
METHODS: 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.
RESULTS: 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.
CONCLUSION: 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.
METHODS: 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.
RESULTS: 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.
CONCLUSION: 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.