结核分枝杆菌抗原Ag85A(Rv3840c)限制性CD8^+ CTL表位的预测及鉴定

Prediction and Identification of HLA-A0201 Restricted CD8^+ CTL Epitopes in Mycobacterium Tuberculosis Ag85A

目的预测及鉴定新的HLA-A*0201结核分枝杆菌(Mycobacterium tuberculosis,Mtb)抗原Ag85A中的CD8+CTL优势表位。方法应用数据库SYFPEITHI预测可能存在的HLA-A*0201的限制性CD8+CTL表位,并经流式细胞术分析抗原肽与HLA-A*0201的亲和力、经时间荧光分辨法检测结核(TB)患者外周血单个核细胞(PBMCs)对抗原肽的增殖反应,再通过细胞毒实验研究抗原肽诱导的T细胞毒杀伤活性,逐步鉴定Ag85A的HLA-A*0201限制性CD8+CTL表位。结果位于Ag85A氨基酸序列(48-56)的肽表位与HLA-A*0201分子具有较高的亲和力,并能刺激HLA-A*0201阳性结核患者PBMC增殖,诱导产生具有特异性杀伤活性的CTL。结论肽6(GLPVEYLQV,48-56aa)是Mtb抗原Ag85A的HLA-A*0201限制性CD8+CTL的优势表位,这为今后进一步研究结核杆菌,作为设计结核疫苗的候选表位奠定了一些基础。

Objective To identify HLA-A ＊ 0201 restricted CD8 ^＋ CTL epitopes in mycobactefium tuberculosis （Mth） antigen Ag85 A. Methods Firstly, database SYFPEITHI was applied to predict HLA-A ＊ 0201 restricted CD8 ^＋ CTL epitopes in Mtb antigen Ag85A. Secondly, affinity and stability of the predicted peptides with T2 cell were assayed by FACS. Thirdly, cells proliferation and cytotoxieity induced by peptides were investigated by DELFIA cell proliferation assay and DELFIA EUTDA cytotoxicity test respectively. Results Peptide 6 （pep6, GLPVEYLQV, 48 -56aa） had high affinity with HLA-A ＊ 0201 molecules and was able to stimulate PBMCs from PPD ＋ healthy donors to proliferate and induce antigen specific CTLs. Only pep6 could give a positive proliferation response and specific cytotoxicity. Conclusion Pep 6 is an HLA-A ＊ 0201 restricted CD8^＋ CTL epitope of Mth antigen Ag85A, which could be the candidate for tuberculous pepfide based vaccine.