制备HLA-A*0201型NLVPMVATV肽四聚体用于巨细胞病毒特异CTL检测

Preparation of HLA-A * 0201 NLVPMVATV peptide tetramers and application to detect cytomegalovirus specific CTL

目的制备HLA-A*0201型NLVPMVATV肽四聚体,用于检测病人的巨细胞病毒(CMV)特异CTL,指导临床用药。方法工程菌以包涵体的形式表达HLA-A*0201型α链和β2m,对包涵体进行洗涤、变性后,在NLVPMVATV肽存在的情况下,在复性液中加入适量的α链和β2m,共同复性形成单聚体,单聚体浓缩后进行生物素化,再纯化浓缩,与PE标记的链亲和素反应,形成四聚体。结果用制备的四聚体-PE和购买的CD8-FITC能双标记CMV特异CTL,用于流式细胞仪检测。结论成功地制备了四聚体-PE,可用于病人外周血的CMV特异CTL检测,由此可知病人的细胞免疫功能。

Objective HLA-A ＊ 0201 NLVPMVATV peptide tetramers were prepared and applied to detect CMV-specific CTL of patient to guide clinical medication. Methods HLA-A ＊ 0201 α chain and β2m were expressed in the form of inclusion bodies flora engineering bacteria. The inclusion bodies were washed and solubilized in urea buffer. NLVPMVATV peptide, α chain and β2m were added to the refolding buffer, and the monomer was refolded. The mixtures were concentrated and biotinylated. Then the refolded molecules were purified and concentrated. The purified monomer was mixed with phycoerythrin-labeled ExtrAvidin to form tetramers. Results The tetramers-PE and CD8-FITC doubly labeled CMV-specific CTL and then subjected to FACS analysis. The preparation of tetramers is successful. The tetramers can be used to detect CMV-specific CTL in the peripheral blood flora HLA-A ＊ 0201 patients and then the cellular immune function of them can be known.