[Construction of HLA-A2-peptide tetramer and application in HBV/HCV infection].
OBJECTIVE
To construct HBV and HCV-specific HLA-A2-peptide tetramers, and to direct clinical therapy.


METHODS
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.


RESULTS
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 cell as inclusion bodies and the proportion were about 85% and 90%, respectively. The purity of pBV220-HLA-A2-BSP and beta-2M was above 95% analyzed by SDS-PAGE, and the concentration of pBV220-HLA-A2-BSP and beta-2M was about 1.5 g/L and 1.2 g/L, respectively. Using the constructed HLA-A2-peptide tetramer to detect the HBV/HCV-specific CTL, the HBV-specific CD8(+) frequencies were 1.84% and 0.02% - 0.68% of the total CD8(+) T cells in acute and chronic HBV hepatitis, respectively. As an additional control, an HLA-A2/HCV tetramer was tested in the acute and chronic HBV hepatitis. The frequencies never exceeded 0.02% of the total CD8(+) T cell number. Similar low levels of background staining were also detected in the HLA-A2(+) or A2(-) healthy control. The HCV-specific CD8(+) frequency was 0.02 - 0.72% of the total CD8(+) T cells in chronic HCV hepatitis. The same frequencies of control were detected.


CONCLUSIONS
High-efficient expressions of HLA-A0201-BSP and beta-2m proteins lay a good foundation for further expression and purification in prokaryotic system and constructing MHC class I-peptide tetramer complexes to study the function of CTLs. Especially, using these two HBV and HCV-specific tetramer can detect the frequencies of the HBV/HCV-specific CD8(+) T cells directly in vitro.
Piao
Wen-hua
WH
Peking University First Hospital, Beijing 100034, China.
He
Yu
Y
Xi
Hong-li
HL
Sun
Xin-ting
XT
Zhang
Heng-hui
HH
Xu
Jing-hang
JH
Zhao
Hong
H
Xu
Wen-xie
WX
Li
Zai-liu
ZL
Wang
Gui-qiang
GQ
D003001:Cloning, Molecular / Q000379:methods; D005260:Female; D015789:HLA-A2 Antigen / Q000235:genetics* / Q000276:immunology*; D019694:Hepatitis B, Chronic / Q000276:immunology*; D019698:Hepatitis C, Chronic / Q000276:immunology*; D006801:Humans; D008285:Major Histocompatibility Complex / Q000235:genetics / Q000276:immunology; D008297:Male; D010446:Peptide Fragments / Q000276:immunology; D011993:Recombinant Fusion Proteins / Q000235:genetics; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology*; D001613:beta 2-Microglobulin / Q000235:genetics / Q000378:metabolism
D015789:HLA-A2 Antigen; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D001613:beta 2-Microglobulin