Preparation and Identification of HLA-A*1101 Tetramer Loading with Human Cytomegalovirus pp65 Antigen Peptide  

作　　者：Fengyao Li Lihui Xu Qingbing Zha Xiaoyun Chi Qiantao Jia Kianhui He 

机构地区：[1]Institute of Tissue Transplantation and Immunology, College of Life Science and Technology, Jinan University, Guangzhou 510632, China [2]Institute of Bioengineering, College of Life Science and Technology, Jinan University, Guangzhou 510632, China [3]Clinical Experimental Center of the First Affiliated Hospital, Jinan University, Guangzhou 510632, China

出　　处：《Cellular & Molecular Immunology》2007年第2期141-146,共6页中国免疫学杂志（英文版）

基　　金：supported by grants from the National Natural Science Foundation of China(No.30230350,No.30572199 and No.30371651).

摘　　要：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, β2-microglobulin （β2m）, 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 Escherichla coli. Moreover, HLA-A＊1101-BSP protein was refolded in the presence of β2m and an HCMV peptide pp6516.24 （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.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, β2-microglobulin （β2m）, 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 Escherichla coli. Moreover, HLA-A＊1101-BSP protein was refolded in the presence of β2m and an HCMV peptide pp6516.24 （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.

关 键 词：HLA-A＊ 1101 TETRAMER CYTOMEGALOVIRUS CD8^＋T cell EPITOPE 

分 类 号：R392-33[医药卫生—免疫学] R373[医药卫生—基础医学]