【摘要】 目的:分析结核分枝杆菌(Mycobacterium tuberculosis,MTB)E7多肽的T细胞抗原表位特性,克隆及分析E7多肽反应阳性的结核患者HLA-DR等位基因。方法:应用从结核患者末梢血分离的单个核细胞(PBMC),以IFN-γ-ELISPOT和细胞内细胞因子染色分析MTB E7多肽的T细胞抗原表位特性;从E7-ELISPOT阳性反应的PBMC或胸水细胞中扩增、克隆和分析HLA-DRα和β链等位基因。结果:311例患者IFN-γ-E7-ELISPOT检测显示233例阳性(阳性率75%),阳性结果的平均SFC为210个斑点/106细胞,E7和另一多肽(E6多肽)混合检测529例患者则显示492例阳性(阳性率93%),平均SFC为572个斑点/106细胞;E7多肽刺激后,19例患者的PBMC经细胞内细胞因子染色结果显示能产生IFN-γ的CD4+T细胞百分率为0.63±1.30,而5例健康对照者为0.05±0.056,两者有显著性差异(P<0.004);扩增、克隆出了共有的HLA-DRA*0101和15种HLA-DRB1等位基因。结论:E7是一种理想的广谱HLA-DR限制性CD4+T细胞反应性多肽,为深入研究结核患者的HLA-DRB等位基因的分布特点以及制备相应四聚体建立了基础。更多还原

【Abstract】 Objective:To study T cell epitope characteristics of peptide E7 from Mycobacterium tuberculosis(MTB),to clone and analyze HLA-DR alleles of the E7-positive patients with tuberculosis(TB).Methods:The peripheral blood mononuclear cells(PBMC) from TB patients were tested by IFN-γ-ELISPOT and intracellular cytokine staining to study T cell epitope characteristics of peptide E7.HLA-DR alleles from PBMC or inflammatory cells in pleural fluid(PLF) of E7/ELISPOT-positive TB patients were cloned and analyzed by a PCR-based recombination technique.Results:233 of 311 and 492 of 529 patients samples(with positive rate 75% and 93%) appeared positive and mean positive SFC was 210 and 572 dots/106 PBMCs by IFN-γ-E7-ELISPOT and IFN-γ-E7+E6 mix-ELISPOT,respectively.The intracellular cytokine staining detections showed that the percentage of CD4+ T cells producing IFN-γ after E7 stimulation in 19 TB patients samples was 0.63±1.30,compared to 0.05±0.056 in 5 healthy control samples(P<0.004).By amplifying,cloning and sequencing,at least 15 different HLA-DRB alleles and HLA-DRA1*0101 were found in these E7/ELISPOT-positive patients identified.Conclusion:Peptide E7 is an ideal CD4+ T cell-responsive and novel HLA-DR-restricted epitope.Obtained HLA-DRB alleles lay a foundation for the further research of distribution of HLA-DRB alleles in TB patients and preparation of E7/HLA-DR tetramers.更多还原