结核分枝杆菌esxN特异性细胞表位的鉴定及其在肺结核诊断中的应用

Identification and application of Mycobacterium tuberculosis esxN-specific cell epitopes in the diagnosis of pulmonary tuberculosis

目的鉴定结核分枝杆菌早期分泌靶向抗原6(ESAT-6)样蛋白esxN特异性HLA-A~*0201限制性细胞毒性T细胞(CTL)表位并用于肺结核诊断。方法采用SYFPEITHI软件预测esxN中受HLA-A~*0201限制性的CTL表位并合成候选表位;采用主要组织相容性复合体(MHC)-肽复合物稳定性实验检测候选表位与HLA-A~*0201分子结合力;采用酶联免疫斑点实验(ELISPOT)研究候选表位在HLA-A~*0201转基因小鼠中的免疫原性;基于鉴定的CTL表位、ESAT-6和培养滤液蛋白10(CFP-10),采用ELISPOT检测肺结核患者体内表位及蛋白特异性CTL的水平。结果 6条候选CTL表位中,esxN_(15-24)(AMIRAQAASL)和esxN_(48-57)(VACQEFITQL)与HLA-A~*0201分子有较高结合力;候选表位免疫HLA-A~*0201转基因小鼠,esxN_(48-57)诱导产生可分泌γ干扰素(IFN-γ)的T细胞反应;在HLA-A~*0201阳性肺结核患者中检测到esxN_(15-24)、esxN_(48-57)、ESAT-6、CFP-10特异性分泌IFN-γ的T细胞;esxN_(15-24)、esxN_(48-57)诊断肺结核的敏感率与ESAT-6、CFP-10相近。结论从结核分枝杆菌esxN中鉴定出两条HLA-A~*0201限制性CTL表位,其有望用于辅助肺结核病的诊断。

Objective To identify Mycobacterium tuberculosis ESAT-6 protein esxN-specific HLA-A~＊ 0201-restricted CTL epitopes and assess the diagnostic potential of the identified epitopes in pulmonary tuberculosis. Methods The esxN-specific HLA-A ~＊ 0201-restricted CTL epitopes were predicted by the T epitope prediction software SYFPEITHI and further synthesized. The binding affinity of the candidate epitopes for HLA-A~＊ 0201 was detected using MHC-peptide complex stabilization assay. The immunogenicity of candidate epitopes were assessed using ELISPOT in HLA-A~＊ 0201 transgenic mice. Based on identified CTL epitopes, ESAT-6 and culture filtrate protein-10（ CFP-10）, the ELISPOT was performed to detect the frequency of epitope/protein-specific CTL. Results In six CTL epitope candidates we tested,two epitopes,esxN_（15-24）（ AMIRAQAASL） and esxN_（48-57）（ VACQEFITQL）,were found to have a high affinity for HLA-A~＊ 0201. In the HLA-A~＊ 0201 transgenic mice immunized with the epitope candidates,esxN_（48-57） induced T-cell response with a significantly high IFN-γsecretion. The IFN-γ-producing T cells directed to esxN_（15-24） and esxN_（48-57） were found to be correlated with the presence of ESAT-6 and CFP-10 in positive pulmonary tuberculosis patients. The sensitivity of these tests for the esxN_（15-24） and esxN_（48-57） epitopes was similar to that of ESAT-6 and CFP-10. Conclusion Two novel Mycobacterium tuberculosis protein esxN-derived HLA-A~＊ 0201-restricted CTL epitopes have potential for the diagnosis of tuberculosis.