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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。
Objective To predict and screen HLA-A * 0201-restricted CD8 + CTL epitopes of Mycobacterium tuberculosis (Mtb) using bioinformatics software. Methods The standard strain H37Rv and Beijing genotype Mycobacterium tuberculosis were used as the research objects. The genes of up-regulated expression after hypoxia culture were screened by using Mycobacterium tuberculosis genome-wide chip technology. Using SYFPEITHI bioinformatics software to analyze the sequence of the polypeptide encoded by the related genes HLA-A * 0201-restricted CD8 + CTL epitopes that may be present; HLA-A2 binding assays using T2 cells to test the binding affinity of the candidate epitope peptide; the binding stability was tested by HLA-A2 dissociation assay; The HLA-A * 0201-restricted CD8 + CTL epitope was thus screened. RESULTS: Four epitope peptides capable of binding to HLA-A * 0201 were screened from epitope peptides predicted by the SYFPEITHI database. Rv0440-1 (KLQER-LAKL), Rv0440-9 (TLLKGAKEV) , RV0350-6 (VLKGEVKDV) and HLA-A * 0201 (FR> 1.5). The epitope peptides Rv0440-1 and Rv0440-9 were obtained by HLA-A2 dissociation assay HLA-A * 0201 molecules with high stability (DC50> 4h). Conclusions The results of bioinformatics techniques and preliminary screening of binding and dissociation of HLA-A2 showed that Rv0440-1 (KLAGGVAVI) and Rv0440-9 (TLLKGAKEV) were identified as latent phase of Mycobacterium tuberculosis HLA-A * 0201-restricted CD8 + CTL Advantage epitope, but to be further experimental verification.