title: Preparation and identification of HLA-A*1101 tetramer loading with human cytomegalovirus pp65 antigen peptide.
abstract: MHC/peptide tetramer technology has been widely used to study antigen-specific T cells, especially for identifying virus-specific CD8+ T cells in humans. The tetramer molecule is composed of HLA heavy chain, beta2-microglobulin (beta2m), an antigenic peptide, and fluorescent-labeled streptavidin. To further investigate the HLA-A*1101-restricted CD8+ T cell responses against human cytomegalovirus (HCMV), we established an approach to prepare HLA-A*1101 tetramer complexed with a peptide from HCMV. The cDNA encoding HLA-A*1101 heavy chain was cloned and the prokaryotic expression vector for the ectodomain of HLA-A*1101 fused with a BirA substrate peptide (HLA-A*1101-BSP) at its carboxyl terminus was constructed. The fusion protein was highly expressed as inclusion bodies under optimized conditions in Escherichia coli. Moreover, HLA-A*1101-BSP protein was refolded in the presence of beta2m and an HCMV peptide pp65(16-24) (GPISGHVLK, GPI). Soluble HLA-A*1101-GPI monomer was biotinylated and purified to a purity of 95%, which was subsequently combined with streptavidin to form tetramers at a yield of > 80%. The HLA-A*1101-GPI tetramers could bind to virus-specific CD8+ T cells, suggesting soluble HLA-A*1101-GPI tetramers were biologically functional. This study provides the basis for further evaluation of HLA-A*1101-restricted CD8+ T cell responses against HCMV infection.
authors.affiliation: Institute of Tissue Transplantation and Immunology, College of Life Science and Technology, Jinan University, Guangzhou 510632, China.
authors.forename: Fengyao
authors.initials: F
authors.lastname: Li
authors.forename: Lihui
authors.initials: L
authors.lastname: Xu
authors.forename: Qingbing
authors.initials: Q
authors.lastname: Zha
authors.forename: Xiaoyun
authors.initials: X
authors.lastname: Chi
authors.forename: Qiantao
authors.initials: Q
authors.lastname: Jia
authors.forename: Xianhui
authors.initials: X
authors.lastname: He
mesh_terms: D019921:Biotinylation; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology* / Q000821:virology; D019731:Carbon-Nitrogen Ligases / Q000378:metabolism; D002469:Cell Separation / Q000379:methods*; D003001:Cloning, Molecular; D003586:Cytomegalovirus Infections / Q000276:immunology*; D018984:Epitopes, T-Lymphocyte / Q000276:immunology; D029968:Escherichia coli Proteins / Q000378:metabolism; D005434:Flow Cytometry; D015234:HLA-A Antigens / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D059870:HLA-A11 Antigen; D006801:Humans; D009842:Oligopeptides / Q000378:metabolism; D010455:Peptides / Q000235:genetics / Q000378:metabolism; D010750:Phosphoproteins / Q000276:immunology* / Q000378:metabolism; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012097:Repressor Proteins / Q000378:metabolism; D019809:Streptavidin / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism; D014763:Viral Matrix Proteins / Q000276:immunology* / Q000378:metabolism; D001613:beta 2-Microglobulin / Q000378:metabolism
chemical_list: D018984:Epitopes, T-Lymphocyte; D029968:Escherichia coli Proteins; D015234:HLA-A Antigens; C515740:HLA-A*11:01 antigen; D059870:HLA-A11 Antigen; D009842:Oligopeptides; D010455:Peptides; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D014157:Transcription Factors; D014763:Viral Matrix Proteins; D001613:beta 2-Microglobulin; C065418:cytomegalovirus matrix protein 65kDa; D019809:Streptavidin; D019731:Carbon-Nitrogen Ligases; C058125:birA protein, E coli