加载HCMV抗原肽的HLA-A*0201单体及其四聚体制备和鉴定

细胞毒T淋巴细胞(CTL)在控制病原体感染以及抗肿瘤过程中发挥重要作用，因而特异性CTL的检测相当重要；而过去检测CTL的方法都是间接的，最近发展起来的四聚体技术则是直接检测抗原特异性CTL的有效而特异的方法，成为目前研究T细胞免疫应答的关键技术。报道一种简化的四聚体制备程序，利用该程序成功制备加载人巨细胞病毒(HCMV)抗原肽的HLA-A2四聚体，具有特异性结合CTL活性。HLA-A*0201重链基因是通过RT-PCR方法从HLA-A2⁺供者白细胞中克隆，进而以PCR方法构建在羧基端融合生物素化酶BirA底物肽(BSP)的HLA-A*0201(A2)重链胞外区原核表达载体, A2重组蛋白在大肠杆菌中得到高表达，主要以包涵体形式存在。加载抗原肽的可溶性A2单体是A2胞外区在轻链β2微球蛋白和HLA-A2限制性HCMV pp65_495-503抗原肽(NLVPMVATV，NLV)存在时通过稀释法复性获得，以BirA对其进行生物素化，然后以阴离子交换树脂纯化，得到的纯化A2-NLV单体与Streptavidin_PE按4:08比例混合形成四聚体，结合程度在85％以上，流式细胞仪分析显示该四聚体具有与HLA-A2+供者的特异性CTL结合活性。总之，这种简化的四聚体制备程序，不仅有利于该技术的推广，为特异性T细胞免疫研究建立必要的技术平台，而且A2-NLV四聚体在临床监测CMV特异性CTL水平等方面也有应用价值。

Preparation and Characterization of HLA-A * 0201 Monomer and Tetramer Loaded with HCMV Antigenic Peptide

Quantification of cytotoxic T lymphocytes (CTL) is extremely important due to the pivotal role they play in controlling pathogen infection and anti-tumor actions. Previously used methods for detecting specific CTL are usually indirect. In recent years, tetramer technology has been developed to directly visualize antigen-specific CTL efficiently, and become the critical approach in studying T cell immune responses. A simplified procedure for preparing tetramers is reported here in this paper and a tetramer loaded with human cytomegalovirus (HCMV) peptide was successfully obtained using this procedure, which possessed binding activity with specific CTL. The heavy chain of HLA-A * 0201 gene was cloned by RT-PCR from HLA-A2+ donor. An expression vector, encoding the extracellular domain of HLA-A * 0201 heavy chain (A2) fused with a BirA substrate peptide (BSP) at its carboxyl terminus, was constructed by PCR with cloned A2 gene as the template. The A2 heavy chain was expressed in Escherichia coli mostly in the form of inclusion body and purified by washing inclusion body. The monomer of soluble A2 loaded with peptide was reconstructed by dilution from the heavy chain in the presence of light chain beta2-microglobulin and HLA-A2 restricted HCMV pp65(495-503) peptide (NLVPMVATV, NLV). Refolded A2-NLV monomer was biotinylated with a commercial BirA and purified by low pressure anion exchange chromatography on a Q-Sepharose (fast flow) column. The tetramer was then formed by mixing A2-NLV monomer with streptavidin-PE in a ratio of 4:0.8 leading to more than 85% multiplication as revealed by SDS-PADE under non-reducing conditions without boiling the sample. Flow cytometry analysis indicated that this tetramer could bind to specific CTL from HLA-A2+ donor. In conclusion, a simplified procedure is established to prepare HLA-A2 tetramer, which may not only facilitate the application of tetramer technology for studying specific T lymphocyte immune response but A2-NLV itself be applied clinically to monitor CMV-specific CTL in stem cell and organ transplantation.