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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 analyze T cell epitope of Mycobacterium tuberculosis (MTB) E7 polypeptide and to clone and analyze the HLA-DR allele of E7 polypeptide-positive tuberculosis patients. Methods: Mononuclear cells (PBMCs) isolated from the peripheral blood of patients with tuberculosis were used to analyze the T cell epitope of MTB E7 polypeptide by IFN-γ-ELISPOT staining and intracellular cytokine staining. From PBMCs with positive E7-ELISPOT or The pleural fluid cells were expanded, cloned and analyzed for HLA-DRα and β-chain alleles. RESULTS: Of the 311 patients, IFN-γ-E7-ELISPOT showed a positive result of 233 (positive rate 75%). The average SFC of the positive results was 210 spots / 106 cells. The E7 and another polypeptide (E6 polypeptide) 492 cases were positive (positive rate was 93%), and the average SFC was 572 spots / 106 cells. After stimulation with E7 peptide, the cytotoxicity of PBMC from 19 patients showed that IFN-γ-producing CD4 + T cell percentage was 0.63 ± 1.30, while that of 5 healthy controls was 0.05 ± 0.056 (P <0.004). A total of HLA-DRA * 0101 and 15 HLA-DRB1 Alleles. Conclusion: E7 is an ideal broad-spectrum HLA-DR-restricted CD4 + T cell reactive polypeptide, which provides a basis for further study on the distribution of HLA-DRB alleles and preparation of corresponding tetramer in tuberculosis patients.