【摘要】 目的采用生物信息学软件预测并筛选结核分枝杆菌(Mycobacterium tuberculosis,Mtb)HLA-A*0201限制性CD8+CTL表位。方法以标准株H37Rv和北京基因型结核分枝杆菌为研究对象,利用结核分枝杆菌全基因组芯片技术筛选缺氧培养后表达上调的基因,应用SYFPEITHI生物信息学软件分析相关基因编码的多肽序列中可能存在的HLA-A*0201限制性CD8+CTL表位;利用T2细胞进行HLA-A2结合试验,检测候选表位肽的结合亲和性,以HLA-A2解离试验检测其结合稳定性,从而筛选出HLA-A*0201限制性CD8+CTL表位。结果从SYFPEITHI数据库预测到的表位肽中筛选出4条能够结合HLA-A*0201分子的表位肽,通过HLA-A2结合试验得到Rv0440-1(KLQER-LAKL)、Rv0440-9(TLLKGAKEV)、RV0350-6(VLKGEVKDV)与HLA-A*0201分子之间均有较高的亲和性(FR>1.5),通过HLA-A2解离试验得到候选表位肽Rv0440-1和Rv0440-9与HLA-A*0201分子的结合具有较高的稳定性(DC50>4h)。结论经生物信息学技术预测以及HLA-A2结合与解离试验初步筛选出的Rv0440-1(KLAGGVAVI)和Rv0440-9(TLLKGAKEV)是潜伏期结核分枝杆菌HLA-A*0201限制性CD8+CTL的优势表位,但有待进一步实验验证。更多还原

【Abstract】 Objectives To use bioinformatics software to predict and screen HLA-A*0201 restricted CD8+CTL epitopes in Mycobacterium tuberculosis(Mtb).Methods H37Rv,a reference Mtb strain,and the Beijing genotype were studied.The Mtb whole genome array was used to screen genes that were up-regulated under anaerobic cultivation.The online database SYFPEITHI was used to predict potential HLA-A*0201 restricted CD8+CTL epitopes.A T2 cell line was used to determine the affinity and stability of the predicted epitope peptides and HLA-A*0201 was used to screen HLA-A*0201 CD8+CTL epitopes.Results Four potential epitopes were identified from the epitope peptides predicted by the SYFPEITHI database.A T2 binding affinity assay indicated that 3 of the 4 peptides(Rv0440-1,Rv0440-9,and Rv0350-6) had high binding affinity for HLA-A*0201 molecules(FR>1.5).A T2 stabilization assay indicated that Rv0440-1 and Rv0440-9 bound stably to HLA-A*0201 molecules(DC50>4 h).Conclusion Bioinformatics software and an assay of HLA-A2 binding and dissociation identified Rv0440-1(KLAGGVAVI)and Rv0440-9(TLLKGAKEV) as HLA-A*0201 restricted CD8+CTL epitopes of latent-phase Mtb.These results should be further verified by additional experiments.更多还原