[Construction of three HLA-A*0201-peptide tetramers and their preliminary application in detection of vitiligo-specific cytotoxic T lymphocytes].
To construct vitiligo-specific HLA-A*0201-peptide tetramers and to apply the constructed tetramers in detection of vitiligo-specific cytotoxic T lymphocytes (CTL).



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.



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.



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.
Clinical Laboratory，Jinhua Hospital of Zhejiang University，Jinhua 321000，China.
Jun-qi
JQ
Wu
Beijing Kuanto Biotech Co. Ltd，Beijing 100176，China.
Jia
J
Shi
Beijing Kuanto Biotech Co. Ltd，Beijing 100176，China.
Jian-feng
JF
Dong
Department of Dermatology，Fifth Hospital of Jinhua City，Jinhua 321000，China.
Yi-xiu
YX
Jiang
Clinical Laboratory，Jinhua Hospital of Zhejiang University，Jinhua 321000，China.
Sun-hong
SH
Zhuang
School of Medicine，Hangzhou Normal University，Hangzhou 310016，China.
Jun
J
Yang
D019921:Biotinylation; D005434:Flow Cytometry; D015789:HLA-A2 Antigen; D006801:Humans; D010455:Peptides; D013602:T-Lymphocytes, Cytotoxic / Q000166:cytology*; D014820:Vitiligo / Q000175:diagnosis* / Q000276:immunology
C435939:HLA-A*02:01 antigen; D015789:HLA-A2 Antigen; D010455:Peptides