肝素酶HLA-A2限制性CTL表位肽的筛选及其活性鉴定

Selection and identification of HLA-A2-restricted CTL epitopes within heparanase

目的预测并鉴定新的人乙酰肝素酶（Hpa）抗原的HLA-A2限制性CTL表位，为恶性肿瘤多肽疫苗的免疫治疗提供依据。方法采用SYFPEITHI和BIMAS软件预测方法，对肝素酶HLA—A2限制性CTL表位进行预测，合成候选表位肽；利用1、2细胞特点，对合成的候选肽与HLA—A2分子进行亲和力分析；利用乳酸脱氢酶释放试验检测待检肽特异性CTL诱导活性；利用ELISPOT检测T细胞活性。结果在所筛选的6条候选CTL表位肽中，Hpa（310～318）FLNPDVLDI与HLA—A2分子具有高亲和力，在体外可有效诱导肝素酶特异性CTL的产生，对肝素酶阳性且HLA—A2限制性的HCC—LM6肝癌细胞及SW-480结肠癌细胞具有明显的杀伤效应，且能有效诱导IFN-1分泌，增强免疫活性。结论首次发现Hpa（310～318）FLNPDVLDI可能是肿瘤肝素酶抗原的HLA-A2限制性CTL表位。

Objective To select and-identify＇human leukocyte antigen （HLA）-A2-restricted T- cell epitope within heparanase （ Hpa）, and to provide academic bases for peptide-based immunotherapy with malignant tumor. Methods The Hpa sequence was scanned for prediction of the immunogenic peptides- based CTL epitopes using the HLA-binding prediction software SYFPEITHI and BIMAS. Then the affinity of candidate epitopes to HLA-A2 was evaluated by T2 binding test. Activation of T cell was assessed by ELIS- POT and cytotoxictiy by lactate dehydrogenase （ LDH ） release assay. Results Of the six predicted nonapeptides, Hpa（310-318, FLNPDVLDI） showed high affinity of binding to HLA-A2 and led to IFN-γsecretion in vitro. Furthermore, Hpa （310-318） FLNPDVLDI could induce PBMC from a HLA-A2 positive healthy donor to lyse specifically HCC-LM6 and SW-480 cells which expressing both Hpa and HLA-A2. Conclusion The nona-peptide Hpa（310-318）FLNPDVLDI may be an HLA-A2-restricted CTL epitope, which is capable of inducing Hpa-specific CTL in vitro.