【摘要】 目的体外鉴定结核分枝杆菌(Mycobacterium tuberculosis,Mtb)Rv0440蛋白序列中表位肽362-370aa和369-377aa的HLA-A*0201限制性CD8+CTL表位的免疫原性,为基于表位的结核疫苗研究提供实验依据。方法根据T2细胞HLA-A*0201分子与多肽结合力分析实验结果,选取结核分枝杆菌Rv0440蛋白质氨基酸序列中对HLA-A*0201分子高亲合力的Rv0440-1(362-370aa,KLQERLAKL)和Rv0440-2(369-377aa,KLAGGVAVI)作为候选表位肽。用候选表位肽刺激PPD(+++)健康志愿者外周血单个核细胞(peripheral blood mononuclear cells,PBMC)检测细胞分泌IFN-γ的水平。用候选表位肽诱导特异性CTL细胞,检测特异性CTL细胞对负载表位肽的T2细胞的杀伤活性,观察Rv0440-1和Rv0440-2的HLA-A*0201限制性CD8+CTL表位的免疫原性。结果 ELISPOT实验结果显示,表位肽Rv0440-1能够明显诱导HLA-A*0201(+)、PPD(+++)健康志愿者PBMC分泌IFN-γ(P<0.05);且表位肽Rv0440-1负载DC诱导的CTL在效靶比为10∶1时对负载相应表位肽的T2细胞的特异性杀伤活性高于对照组(P<0.05);与对照组相比,表位肽Rv0440-2没有诱导能力。结论表位肽Rv0440-1(362-370aa,KLQERLAKL)具有良好的免疫原性,是有效的结核分枝杆菌的HLA-A*0201限制性CTL表位。更多还原

【Abstract】 The immunogenicity of HLA-A*0201restricted CD8+CTL epitopes 362-370aa and 369-377aa in Mycobacterium tuberculosis(Mtb)Rv0440protein were identified in this study,so as to provide evidence for epitope-based study for tuberculosis(TB)vaccine.T2cell line was used to assay the affinity between the predicted peptides and HLA-A*0201molecules.Based on peptides with high binding affinity to HLA-A*0201molecules,Rv0440-1(362-370aa,KLQERLAKL)and Rv0440-2(369-377aa,KLAGGVAVI)were chosen to be the candidate epitopes.The secreting IFN-γrelease of peripheral blood mononuclear cells(PBMC)was investigated.The specific CTLs were induced from PBMC of HLA-A*0201(+)and PPD(+++)in healthy donors by the candidate peptides.In vitro cytotoxicity of peptide-induced CTL was determined to screen HLA-A*0201restricted CD8+CTL epitopes from those candidates.The result showed that Rv0440-1significantly induced the HLA-A*0201(+)and PPD(+++)of donors’ PBMC to secrete IFN-γby ELISPOT(P<0.05).And when the proportion of effective cells to target cells(E:T)was 10∶1,CTL induced by dendritic cells(DC)loaded with Rv0440-1had higher cytotoxicity to T2target cells(P<0.05),while Rv0440-2had no significant inducing activity.So the conclusion is Rv0440-1(362-370aa,KLQERLAKL)might be the effective HLA-A*0201restricted CTL epitope.更多还原