Generation, Identification Of Tetrameric Peptide -HLA Complexes In Vitro

Human leukocyte antigen(HLA) class I molecules are cell surface glycoproteins encoded by the major histocompatibility complex(MHC) cluster of genes whose main biological function is to bind antigenic peptides(epitopes) and present them to cytotoxic T lymphocytes(CTL). Quantitiative analyses of antigen-specific T cell populations have provided important information on the natural course of immune responses. In the present study,we constructed HLA-peptide tetramers by DNA recombinant techniques, which provides a powerful tool for assessing fundamental aspects of T-cell immunity and should also be of great help in therapeutic follow-up and in particular in evaluating T-cell vaccination strategies. The whole study consists of two sections as the following:HLA-A*0201 typing by PCR-SSP and by sequencing.This study develops a comprehensive easy method to perform HLA-A*0201 typing from the population group. Two pair of primers for HLA-A2 group specific are designed and nested PCR can be used to amplify HLA-A2 gene. As confirmed by DNA sequencing of exon2 and exon3, this allele is identical to HLA-A*02011-02016. This findings of this study have implications for the selection of HLA-A*0201 by virtue of its simplicity and cheapness,it was useful in basic research and clinical research.2. Generation of tetrameric peptide-HLA complexes in vitro.The HLA-A2 gene was amplified by PCR, and was added a 15-amino acid substrate peptide for BirA-dependent biotinylation to the COOH-terminus of heavy chain. Both the heavy chain and the β2m were over-expressed in Escherichia coli as insoluble proteins respectively. Inclusion bodies were dissolved in urea, the refolding was performed by diluting the subunits(heavy chain and β2m)in a large volume of refolding buffer in the presence of antigenic peptide CEA694-702(GVLVGVALI). The HLA complex was further purified by anion exchange agents, fractions containing the complexes were collected for further enzymatic biotinylation. The HLA tetramers were prepared by mixing the biotinylated with fluorescent streptavidin at a molar ratio of 4:1,also,we finished preliminary identification of the structure of tetrameric peptide-HLA complexes in vitro.The ratio of biotinylation had attended 90%.The soluble tetramer provides a powerful tool for assessing fundamental aspects of T-cell immunity.