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目的设计并鉴定具有负调致病细胞毒性T细胞(cytotoxic Tlymphocyte,CTL)免疫应答功能的改造肽配体(al-tered peptide ligand,APL),为基于胰岛素自身抗原的I型糖尿病特异性免疫治疗奠定基础。方法利用InSilico技术模拟构建TCR-pMHC(TCR-HLA-A*0201-mInsB5-14)复合物三维结构,根据结构选择改造位点P6;通过保守/不保守单个氨基酸虚拟替换得到若干候选APL;通过自由能变化计算、相对亲和力检测、细胞因子分泌水平检测等体外功能实验,初步鉴定具有拮抗效应的APL。结果筛选得到的APL mInsB5-14H6F(H→F)与天然肽mInsB5-14空间结构相似,能与HLA-A*0201分子结合,其HLA-A*0201分子的相对亲和力与mInsB5-14相近。该APL作用下mInsB5-14刺激CD8+T细胞分泌IFN-γ的水平明显低于天然肽单独刺激下特异性CTL分泌IFN-γ的水平(P<0.05)。结论基于天然自身抗原表位mInsB5-14改造得到的mInsB5-14H6F是HLA-A*0201限制性的APL,该APL通过降低致病CTL特异性分泌IFN-γ的水平,可能诱导HLA-A*0201转基因NOD小鼠的自身耐受,从而为I型糖尿病治疗性疫苗的研发奠定了基础。
OBJECTIVE: To design and identify al-tered peptide ligand (APL) with the negative immune response function of cytotoxic T lymphocyte (CTL), which is a type I diabetes-specific immunotherapy based on insulin autoantigen Lay the foundation. Methods The three-dimensional structure of TCR-pMHC (TCR-HLA-A * 0201-mInsB5-14) complex was constructed by InSilico technique and site P6 was selected based on structural selection. Several candidate APLs were obtained by the virtual substitution of conservative / Free energy change calculation, relative affinity test, cytokine secretion test in vitro and other in vitro experiments to initially identify APL with antagonistic effect. Results The mSIB5-14H6F (H → F) was similar to mlsB5-14 in its natural structure and could bind to HLA-A * 0201. The relative affinity of mInsB5-14H6F was similar to that of mInsB5-14. The level of IFN-γ secreted by mInsB5-14 stimulated CD8 + T cells under the action of APL was significantly lower than that of IFN-γ secreted by specific CTL stimulated by native peptide alone (P <0.05). Conclusion mInsB5-14H6F, which was modified based on the native self-epitope mInsB5-14, is an HLA-A * 0201-restricted APL that may induce HLA-A * 0201 by decreasing the level of IFN- [gamma] secreted by the pathogenic CTL The self-tolerance of transgenic NOD mice lays the foundation for the development of a therapeutic vaccine for type I diabetes.