E7多肽的抗原表位特性与多肽反应阳性结核患者的HLA-DR等位基因分析  

Analysis of epitope characteristics of peptide E7 and HLA-DR alleles of the E7-positive patients with tuberculosis

作　　者：方毅敏[1] 任亮亮[2] 李研[2] 冯永忠[1] 彭毅[2] 董涛[2] 詹能勇[2,3] 黄宇虹[2] 程俊[2] 马志明[1] 赖小敏[2] 

机构地区：[1]广州市胸科医院,广州510095 [2]中山大学中山医学院微生物学教研室,热带病防治研究教育部重点实验室,广东省重大传染病预防和控制技术研究中心,广州510080 [3]深圳市第三人民医院,深圳518020

出　　处：《中国免疫学杂志》2011年第2期163-166,171,共5页Chinese Journal of Immunology

基　　金：国家自然科学基金重点项目(30430660);传染病重大专项(2008ZX-10003-007、2008ZX10003-012、2009ZX10003-018);广东省科技计划项目(73107)资助

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。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.

关 键 词：结核 E7多肽 HLA-DR 

分 类 号：R392.11[医药卫生—免疫学] R378.91[医药卫生—基础医学]