结核分枝杆菌Rv0440 CTL表位肽的免疫原性研究

Immunogenicity of CTL epitopes in Mycobacterium tuberculosis Rv0440 protein

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。

The immunogenicity of HLA-A ＊ 0201 restricted CD8＋ CTL epitopes 362-370 aa and 369-377 aa in Mycobacterium tuberculosis （Mtb） Rv0440 protein were identified in this study,so as to provide evidence for epitope-based study for tuberculosis（TB） vaccine.T2 cell line was used to assay the affinity between the predicted peptides and HLA-A ＊ 0201 mole-cules.Based on peptides with high binding affinity to HLA-A ＊ 0201 molecules,Rv0440 1（362-370 aa,KLQERLAKL） and Rv0440-2（369-377 aa,KLAGGVAVI） were chosen to be the candidate epitopes.The secreting IFN-γ release 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 ＊ 0201 restricted CD8-CTL epitopes from those candidates.The result showed that Rv0440-1 significantly 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 Rv0440-1 had higher cytotoxicity to T2 target cells （P〈0.05）,while Rv0440-2 had no significant inducing activity.So the conclusion is Rv0440-1（362-370 aa,KLQERLAKL） might be the effective HLA A ＊ 0201 restricted CTL epitope.