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。
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.
出处
《中国人兽共患病学报》
CAS
CSCD
北大核心
2014年第5期489-493,共5页
Chinese Journal of Zoonoses
基金
"艾滋病和病毒性肝炎等重大传染病防治"科技重大专项(No.2009ZX1004-305)~~
关键词
结核分枝杆菌
表位肽
CD8+CTL
特异性杀伤活性
Mycobacterium tuberculosis
epitope peptides
CD8 + cytotoxic T-lymphocytes
specific cytotoxicity