【作者】 吕虹；

【导师】 祁元明； 高艳锋；

【作者基本信息】 郑州大学 ， 细胞生物学， 2010， 硕士

【摘要】 结核病(Tuberculosis, TB)是由结核杆菌(Mycobacterium tuberculosis,Mtb)引起的一种慢性传染病,目前仍然是导致传染病发病率和死亡率较高的疾病之一。尤其是近年来,由于人体免疫缺陷病毒双重感染的出现而造成个体机会性感染愈发严重。目前唯一可用的用于结核病预防的疫苗是卡介苗(Bacillus Calmette-Guerin, BCG),但其在预防成年人患肺结核的有效性方面受到了限制,并且伴随着结核杆菌多重耐药菌株的出现,人们迫切需要研制新型的抗结核疫苗。由CD4+辅助性T(Th1)细胞和CD8+毒性T淋巴细胞(CTL)所介导的细胞免疫反应在抗结核感染保护性免疫中发挥着主导作用。结核新型疫苗的研究依赖于能被CD8+T细胞识别从而产生IFN-γ而发挥杀伤效应的结核抗原及其相应表位的鉴定。分泌蛋白以及细胞壁蛋白是结核杆菌主要的免疫保护抗原,并且位于Mtb基因组和BCG差别区(RD区)内的分泌蛋白由于其在BCG中的缺失,逐渐成为疫苗候选抗原的热点。优势抗原表位的鉴定,对发展更有效的结核病治疗性多表位肽疫苗具有重要的意义,并且能够为基础研究提供理论基础。结核杆菌分泌蛋白CFP21 (culture filtrate proteins 21,培养滤液蛋白21)位于RD2区,具有高度的免疫原性。CFP21能够诱导结核感染结核患者产生T细胞增殖反应以及释放高水平IFN-γ,发挥细胞杀伤作用,是抗结核疫苗设计的理想候选抗原。CFP21 HLA-A*0201/03限制性表位的鉴定与改造主要通过以下工作来完成：我们首先运用在线数据库,以SYFPEITHI初步预测,结合BIMAS和NetCTL 1.2在线分析,初步筛选出一系列的天然表位肽。已有相关文献报道,通过对抗原表位锚定位点的氨基酸替换,能够增强表位与HLA-Ⅰ分子的结合力。因此,我们通过将天然表位,按1Y、2L和/或9L进行氨基酸置换,获得改造肽,利用数据库再次分析,以期寻求结合力更高的表位。综合分析预测结果,选取在至少两种数据库中评分结果位于前10的表位肽,分别是4条母体肽p5 (SLVRIVGVV)、p13 (VVATTLALV)、p134 (AVADHVAAV)、p189 (NIMAHVSYV)以及3条改造肽p189-1Y2L9L(YLMAHVSYL)、p134-1Y2L (YLADHVAAV)、p134-1Y2L9L (YLADHVAAL)。对已经筛选出来的表位,我们采用标准Fmoc方案合成多肽,产物经反向高效液相色谱(RP-HPLC)分析、纯化,获得了纯度高于95%的九肽产物。经质谱测定,分子量所得测定值与理论值相符合。通过内源性抗原提呈途径中所必需的抗原多肽转运蛋白(TAP)缺陷的T2细胞结合力以及肽/HLA稳定性实验,对初步筛选的表位进行验证。结果显示,相对于母体肽p134,改造肽p134-1Y2L、p134-1Y2L9L与HLA分子具有更高的结合力与稳定性。通过结合力及稳定性的初步筛选,在7条候选肽中,只有母体肽p134以及相应的两条改造肽,用于后续ELISPOT以及细胞毒杀伤作用的T细胞免疫活性检测。通过HLA-A*0201+PPD+/HLA-A*03+PPD+健康供者外周血单核细胞(PBMCs)诱导出能够分泌IFN-γ的细胞数的分析以及特异性CTL的LDH杀伤活性检测显示,母体肽p134以及两条改造肽p134-1Y2L和p134-1Y2L9L都能够诱导T细胞反应,但只有p134诱导出的CTL对靶细胞具有明显的杀伤效应。通过上述的工作,我们证明,CFP21134-142 (AVADHVAAV),是CFP21潜在的免疫优势HLA-A*0201/03限制性表位。本课题鉴定出一个源于结核杆菌分泌蛋白CFP21的HLA-A*0201/03限制性表位—p134 (AVADHVAAV)。该表位能有效激发HLA-A*0201和HLA-A*03限制性CTL的免疫应答,且为一种多等位基因广谱CTL表位,能够为结核病的疫苗设计及免疫治疗奠定基础。更多还原

【Abstract】 Tuberculosis, caused by the bacterium, Mycobacterium tuberculosis (Mtb), remains a leading cause of infectious disease morbidity and mortality, which has emerged as a major opportunistic infection in individuals with HIV/AIDS. The only tuberculosis (TB) vaccine currently available is the attenuated Mycobacterium bovis strain Bacillus Calmette-Guerin (BCG), which is ineffective on preventing pulmonary TB in adults. The uncertain efficacy of the BCG vaccine for TB in adults, and the emergence of extensively drug resistant Mtb strains have further enhanced the urgency of the development of novel TB vaccines.The cellular arm of the immune response mediated by CD4+ type 1 helper T (Th1) and CD8+ cytotoxic T lymphocytes (CTL) has been determined to be a pivotal component of protective immunity against Mtb. In order to develop new vaccines against tuberculosis, attention has been directed towards the identification of antigens/epitopes recognized by CD8+ T cells secreting interferon-γ(IFN-γ). Secreted and surface-exposed cell wall proteins seem to play a pivotal role in the induction of protective cellular immunity against TB. Recently, secreted proteins encoded by different regions of difference (RDs) of the Mtb genome have been considered as attractive candidates owing to their absence in most BCG strains. Detailed knowledge of the epitopes recognized by immune responses can aid in peptide-based anti-TB vaccines development, and provide important tools for basic research.A major secreted protein, CFP21, locating in RD2 was demonstrated to be an immunodominant antigen by elevating T cell proliferate response and production of cytokines such as IFN-y. CFP21 could also induce an optimum level of cytotoxic T cell activity. These findings suggested that CFP21 could be used as an attractive candidate antigen.In the present study, to identify novel CFP21-derived HLA-A*0201/03 restricted epitopes, a series of native peptides were predicted with various prediction programs including SYFPEITHI, BIMAS and NetCTL 1.2. Recent studies suggested that modification of amino acids at the anchor position of epitopes could result in enhancing the binding affinity to HLA-I molecules. Here, we designed analogues of the native peptides by altering the native peptides with tyrosine at position 1 (1Y), leucine at position 2 (2L) and/or position 9 (9L). Based on the three prediction programs, four native peptides, p5 (SLVRIVGVV), p13 (VVATTLALV), p134 (AVADHVAAV), p189 (NIMAHVSYV) and three of their analogues, p189-1Y2L9L (YLMAHVSYL), p134-1Y2L (YLADHVAAV), p134-1Y2L9L (YLADHVAAL) with prediction scores in the top 10 detected by at least two of the programs were selected and synthesized for further study. Peptides were synthesized by standard solid phase Fmoc strategy and were purified to more than 95% purity by reverse phase high performance liquid chromatography (RP-HPLC). Their molecular weights were confirmed by electro-spray ionization mass spectrometry (ESI-MS).To evaluate the binding affinity of these peptides to HLA-A*0201/03 molecules and the stability of the peptide/HLA-A complexes in vitro, TAP-deficient T2 cells were used. In concordance with the binding affinities, these two analogues p134-1Y2L and p134-1Y2L9L showed more potent binding stabilities than the native peptide, p134. Based on the results of the T2 binding assay and peptide/HLA stability assay, among the seven peptides studied, only p134 and its two analogues were selected to investigate their ability to induce T cell response by using ELISPOT and cytotoxicity assay. Subsequent IFN-γrelease and LDH release assays by using PBMCs from HLA-A*0201+/03+ PPD+ healthy donors showed that the native peptide p134 and its two analogues p134-1Y2L and p134-1Y2L9L could induce potent T cell response, but only p134-induced CTLs could potently lyse the peptide-loaded target cells. All these results demonstrated that the amino acid sequence of 134-142 (AVADHVAAV) in CFP21 could be an immunogenic CTL epitope which could be both processed and presented endogenously via HLA-A*0201/03.In the present study, a novel HLA-A*0201/03 restricted T cell epitope, p134 (AVADHVAAV), was identified in a secreted protein of Mtb, CFP21. The potent T cell response activity and the potential to be a broad-spectrum epitope made p134 a promising epitope for vaccination and immunotherapy against TB.更多还原