Abstract
Tetramer analysis is a novel technique in immunological research that has dramatically changed our knowledge of the immune response to pathogens, tumors and autoimmune disease. Through the formation of major histocompatibility complex (MHC)-peptide tetrameric complexes, it can provide accurate counts of antigen-specific T-cells and it allows their phenotypical and functional analysis. The tetramer is composed of the human leukocyte antigen (HLA) heavy chain, β-2 microglobulin (β-2m), the nominal peptide, and streptavidin. The HLA heavy chain and the β-2m are expressed in Escherichia coli. But up to now, all laboratories have been expressing these two proteins by using isopropyl β- d-thiogalactopyranoside IPTG [1–6]. IPTG is very expensive, and it is tedious and laborious to induce expression protein. So it is difficult to scale up to express the objective protein [7]. To address this problem, extracellular fractions of HLA-A0201 and β-2m (absent signal peptide) genes were cloned from peripheral blood mononuclear cells (PBMCs) by RT-PCR. DNA coding for a Gly-Ser linker and a BSP (15-amino acid substrate peptide for BirA-dependent biotinylation) was added to the COOH-terminus of the extracellular fraction of HLA-A0201 by PCR, using an HLA-A0201 as the template. Then the HLA-A0201-BSP and β-2m genes were cloned into pBV220 vector and expressed, respectively. The expressed proteins were purified and detected by ELISA and Western blot analyses. High-efficient expressions of HLA-A0201-BSP and β-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.
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