title: [Construction of HLA-A2-peptide tetramer and application in HBV/HCV infection].
abstract: 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.
authors.affiliation: Peking University First Hospital, Beijing 100034, China.
authors.forename: Wen-hua
authors.initials: WH
authors.lastname: Piao
authors.forename: Yu
authors.initials: Y
authors.lastname: He
authors.forename: Hong-li
authors.initials: HL
authors.lastname: Xi
authors.forename: Xin-ting
authors.initials: XT
authors.lastname: Sun
authors.forename: Heng-hui
authors.initials: HH
authors.lastname: Zhang
authors.forename: Jing-hang
authors.initials: JH
authors.lastname: Xu
authors.forename: Hong
authors.initials: H
authors.lastname: Zhao
authors.forename: Wen-xie
authors.initials: WX
authors.lastname: Xu
authors.forename: Zai-liu
authors.initials: ZL
authors.lastname: Li
authors.forename: Gui-qiang
authors.initials: GQ
authors.lastname: Wang
mesh_terms: 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
chemical_list: D015789:HLA-A2 Antigen; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D001613:beta 2-Microglobulin