[Construction and primary application of two HBV-HLA-A*2402-peptide tetramers].
OBJECTIVE
To construct two soluble HLA-A*2402-peptide tetramers and detect the HBV-specific cytotoxic T lymphocytes (CTLs) with the constructed HLA-A*2402-peptide tetramers.


METHODS
After proteins HLA-A*2402-BSP and beta2m were made an effective prokanyotical expression, the purified proteins were refolded into HLA-A*2402-beta2m-peptide complexes in the presence of two antigenic peptides (Hepatitis B virus Pol756-764 or Core117-125) with dilution method. Then the complexes were biotinylated by BirA enzyme and purified by gel-filtration chromatography. The tetramers were generated by mixing the complex with PE-Streptavidin in the molar ratio of 5:1. FCM can and cell quest software were used to analyze the stained PBMCs.


RESULTS
Two biotinylated HBV-HLA-A*2402-peptide complexes were identified by Western blot and they were shown to have natural conformation by Dot-ELISA and ELISA.


CONCLUSIONS
The constructed HLA-A*2402-peptide tetramers can detect the HBV-specific CTLs in the patients with self-limited acute HBV infection.
Yang
Xin-Xing
XX
Division of Clinical Immunology, Tongji Hospital, Tongji Medical College, Huazhong Umiversity of Science and Technology, Wuhan, China. glp5177@126.com
Hao
You-Hua
YH
Song
Na
N
Chen
Ling
L
Liu
Zhi
Z
Yang
Dong-Liang
DL
D000818:Animals; D019921:Biotinylation; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D015234:HLA-A Antigens / Q000737:chemistry* / Q000276:immunology* / Q000302:isolation & purification / Q000378:metabolism; D059871:HLA-A24 Antigen; D006515:Hepatitis B virus / Q000276:immunology*; D006801:Humans; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000276:immunology* / Q000378:metabolism*; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology
D015234:HLA-A Antigens; C438506:HLA-A*24:02 antigen; D059871:HLA-A24 Antigen; D010455:Peptides