HLA-A2肽四聚体的构建及其在乙、丙型肝炎中的初步应用

目的　分别构建乙型肝炎病毒 (HBV)和丙型肝炎病毒 (HCV)特异的HLA A2肽四聚体 ,为检测乙、丙型肝炎患者体内特异性的细胞毒T细胞 (CTL)提供直接、有效的方法 ,指导临床用药。方法　分别构建含有HLA A2 BSP(含有 15个可供识别生物素化的氨基酸位点 )和 β2微球蛋白(β 2m)基因的原核表达载体 ,并进行表达、复性、鉴定及纯化。再分别将HBV和HCV特异性短肽与HLA A2 BSP和 β 2m蛋白在体外进行耦合 ,该复合物经纯化浓缩后进行生物素化。生物素化的产物经纯化浓缩后形成单体 ,此单体再与藻红蛋白标记的链霉亲和素按一定比例耦合构建成四聚体 ,最后进行流式细胞仪检测。结果　获得了高效、稳定的pBV2 2 0 HLA A2 BSP和 pBV2 2 0 β 2m原核表达载体 ,表达量分别占菌体的 4 6 %和 2 6 %左右 ;纯度达 90 %以上。构建的四聚体可成功的检测急、慢性乙型和丙型肝炎患者中特异性的CTL ,急性HBV感染时CTL占 1 84 % ,慢性HBV感染时CTL占0 0 2 %～ 0 6 8% ,慢性HCV感染的CTL占 0 0 2～ 0 72 %。结论　高效、稳定的pBV2 2 0 HLA A2 BSP和 pBV2 2 0 β 2m原核表达载体大量的表达可用于构建各种肽特异性的四聚体复合物 ;在体外构建的MHC Ⅰ类分子四聚体 ,为特异性细胞毒T细胞的检测提供有效的工具。

Construction of HLA-A2-peptide tetramer and application in HBV/HCV infection

OBJECTIVE: To construct HBV and HCV-specific HLA-A2-peptide tetramers, and to direct clinical therapy. METHODS: Recombinant class I HLA-A2 heavy chains and beta-2 M were produced in Escherichia coli cells transformed with pBV220 vectors. Only the extracellular domain of class I heavy chain was expressed, following modification by replacement of the C-terminal domain with a substrate sequence for BirA biotinylation. HLA-A2-BSP was folded in the presence of beta-2 microglobulin and a specific peptide to form a peptide-MHC complex. The MHC complexes were biotinylated using purified BirA enzyme. Biotinylated MHC-peptide complexes were purified. Tetramers were generated by mixing biotinylated protein complex with streptavidin-PE at a molar ratio of 4:1. Then analysis of stained PBMCs was performed using FACScan and CellQuest software. RESULTS: The expression levels of pBV220-HLA-A2-BSP and beta-2M were 46% and 48% of total bacterial proteins estimated from SDS - PAGE, respectively. And they were mainly located in the insoluble fraction of the cell as inclusion bodies and the proportion were about 85% and 90%, respectively. The purity of pBV220-HLA-A2-BSP and beta-2M was above 95% analyzed by SDS-PAGE, and the concentration of pBV220-HLA-A2-BSP and beta-2M was about 1.5 g/L and 1.2 g/L, respectively. Using the constructed HLA-A2-peptide tetramer to detect the HBV/HCV-specific CTL, the HBV-specific CD8(+) frequencies were 1.84% and 0.02% - 0.68% of the total CD8(+) T cells in acute and chronic HBV hepatitis, respectively. As an additional control, an HLA-A2/HCV tetramer was tested in the acute and chronic HBV hepatitis. The frequencies never exceeded 0.02% of the total CD8(+) T cell number. Similar low levels of background staining were also detected in the HLA-A2(+) or A2(-) healthy control. The HCV-specific CD8(+) frequency was 0.02 - 0.72% of the total CD8(+) T cells in chronic HCV hepatitis. The same frequencies of control were detected. CONCLUSION: High-efficient expressions of HLA-A0201-BSP and beta-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. Especially, using these two HBV and HCV-specific tetramer can detect the frequencies of the HBV/HCV-specific CD8(+) T cells directly in vitro.