T细胞表位在抗病毒的T细胞免疫中发挥核心作用,目前已在多种病原微生物的蛋白序列上发现存在T细胞表位的聚集现象。本文建立了一套功能学与结构学结合的策略鉴定病原体上细胞毒性T细胞(Cytotoxic T Lymphocyte,CTL)表位富集区的方法,并以严重急性呼吸道综合征(SARS)相关冠状病毒(SARS-CoV)的M蛋白为例,成功地鉴定了一个HLA-A2限制性的表位富集区。首先通过生物信息学的方法预测并合成M蛋白跨膜区的HLA-A2潜在结合多肽,通过体外复性实验和T2细胞结合实验验证多肽与HLA-A2的结合力;然后在HLA-A2.1/Kb转基因小鼠中检测这些多肽的免疫原性;最后通过X射线衍射技术,成功解析了其中一条多肽与HLA-A^*0201的复合物结构,其结构显示该多肽具有典型的HLA-A^*0201表位的结构特点,但却呈现出与以往鉴定多肽不同的构象和锚定残基。本文对于理解机体对SARS-CoV等病原体产生的T细胞免疫反应,以及为更广泛的人群设计T细胞疫苗具有重要意义。

T cell epitopes play an important role in the anti-virus specific T cell immunity. Currently, the phenomenon of T cell epitope clustering is reported among different pathogenic microbes. Here, a strategy is established to identify Cytotoxic T Lymphocyte (CTL) epitope clusters region with the combined functional and structural methods. And by using this strategy, a HLA-A2-restricted epitope cluster in SARS-CoV M protein is successfully defined. Firstly, the computer-assisted algorithm was utilized to predict a series of potential HLA-A2 binding peptides around the sequences of two previous identified HLA-A2 epitopes Mn2 and Md3 in the transmembrane domain of SARS-CoV M protein. The HLA-A2 binding affinities of these peptides were accessed through in vitro refolding assay and T2 cell binding assay. Consequently, antigenicity of the peptides was confirmed by ELISPOT by using transgenic mice immunized with peptide Mn2 or DNA vaccine of M protein. Furthermore, the crystal structure of HLA-A^*0201 complexed with peptide Md3-C9 was determined. And the structure shows that the peptide is presented in a traditional conformation of HLA-A2 restricted peptides, however is different from the previously-determined structure of Md3. The result is a great help to understand anti-virus T cell responses and might pave a new way for the T cell-related vaccines for a broader population.