目的：鉴定肿瘤抗原PIWIL2的人类白细胞抗原A2（HLA-A2）限制性细胞毒性T淋巴细胞（CTL）表位。方法：首先运用RT-PCR、Western blot方法检测PIWIL2在肿瘤细胞系MCF-7、SW480和HT-29中的表达情况，然后通过BIMAS、RankPep、NetMHC、NetCTL1.2及IEDB软件预测打分来选取PIWIL2的HLA-A2限制性的表位。候选表位肽通过标准的Fmoc化学合成法合成，结合力实验用于检测候选表位与T2A2细胞表面HLA-A2分子的结合能力，ELISPOT实验检测候选表位肽诱导CTL分泌IFN-γ的能力，体外细胞毒性实验检测候选肽诱导CTL的能力。结果：PIWIL2在肿瘤细胞系MCF-7、SW480和HT-29中均有表达。候选肽P485、P493、P965具有中等结合力。 ELISPOT实验结果显示表位肽P485和P965诱导的CTL具有分泌IFN-γ的能力。细胞毒性实验结果显示表位肽P485和P965对MCF-7细胞均有一定的杀伤作用。结论：表位肽P485和P965是优秀的PIWIL2抗原HLA-A2限制性CTL候选表位，可能成为新的抗肿瘤多肽免疫治疗疫苗的候选表位。%AIM: To identify the human leucocyte antigen A2 (HLA-A2) restricted cytotoxic T lymphocyte (CTL) epitopes from tumor antigen PIWIL2.METHODS:RT-PCR and Western blot was used to determine the expres-sion of PIWIL2 in cancer cell lines MCF-7, SW480 and HT-29.HLA-A2 epitopes from PIWIL2 protein were predicted by the software of BIMAS, RankPep, NetMHC, NetCTL1.2 and IEDB.The peptides were synthesized by standard solid-phase methods.The binding affinity of the peptides to HLA-A2 molecules was evaluated by T2 cells binding assay.ELISPOT as-say was used to investigate the levels of IFN-γ.The cytotoxicity assay in vitro was also used to determine the ability of indu-cing T cell response by the peptides.RESULTS:The expression of PIWIL2 was observed in MCF-7, SW480 and HT-29. The candidate peptide P485, P493 and P965 showed moderate affinity toward HLA-A2 molecule.ELISPOT assay showed P485 and P965 induced CTLs of IFN-γrelease form CTLs.The CTLs induced by P485 and P965 lysed the MCF-7 cells. CONCLUSION:The peptides P485 and P965 are excellent HLA-A2 restricted cytotoxic T lymphocyte epitopes from the tumor antigen PIWIL2, which could serve as new candidates towards antitumor peptide vaccines.
展开▼
