胰腺癌高表达抗原MUC4 CTL表位肽的筛选与改造  

Identification and molecular modification of HLA-A2-restricted cytotoxic T-lymphocyte epitopes from pancreatic tumor antigen MUC4

作　　者：范柳笛[1] 刘辉[1] 袁磊[1] 时冉冉[1] 

机构地区：[1]漯河医学高等专科学校医学生物工程重点实验室,河南漯河462002

出　　处：《中国病理生理杂志》2017年第5期811-816,共6页Chinese Journal of Pathophysiology

基　　金：河南省科技厅科技发展计划项目(No.142102310203)

摘　　要：目的:观察胰腺癌高表达抗原黏蛋白4(MUC4)的改造表位是否有HLA-A2限制性抗肿瘤能力。方法:首先运用RT-PCR和Western blot方法检测MUC4在胰腺癌细胞系CAPAN-2和ASPC-1的表达情况。通过Net CTL 1.2、BIMAS、SYFPEITHI和IEDB软件预测打分来选取MUC4的HLA-A2限制性表位;替换MUC4抗原锚定位点氨基酸获得改造肽;候选表位肽的合成方法为标准的Fmoc化学合成法,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细胞毒性T淋巴细胞(CTL)分泌IFN-γ的能力,体外细胞毒实验活性检测候选肽诱导CTL的能力。结果:MUC4在胰腺癌细胞系CAPAN-2和ASPC-1均有表达。P1944-1Y、P1944-2L、P1944-1Y2L、P2004和P2004-1Y9V具有较好的结合力,且P2004-1Y9V、P1944-1Y2L等改造肽与HLA-A2的结合力高于原肽。ELISPOT实验结果显示表位肽P1944、P1944-1Y2L、P2004和P2004-1Y9V诱导的CTL具有分泌IFN-γ的能力。P1944-1Y2L和P2004-1Y9V诱导特异性T细胞免疫分泌的IFN-γ略高于原肽。细胞毒实验结果显示表位P1944、P1944-1Y2L、P2004和P2004-1Y9V对CAPAN-2细胞均有一定的杀伤作用。P1944-1Y2L和P2004-1Y9V特异性CTLs对CAPAN-2细胞杀伤率高于原肽特异性CTLs。结论:MUC4抗原改造表位P1944-1Y2L、P2004-1Y9V与天然表位P1944、P2004相比有更高的HLA-A2分子亲和力,保留了原有的免疫原性,并且改造肽抗肿瘤免疫效应强于天然表位。P1944-1Y2L和P2004-1Y9V是优秀的MUC4抗原的HLAA2限制性CTL候选表位,可以成为新的抗肿瘤多肽免疫治疗疫苗的候选表位。AIM：To observe whether modified epitopes from pancreatic tumor antigen mucin 4 （MUC4） have HLA-A2-restricted antitumor ability.METHODS： RT-PCR and Western blot were used to identify the expression of MUC4 in the pancreatic tumor cell lines CAPAN-2 and ASPC-1. HLA-A2 epitopes from MUC4 protein were predicted by the software of NetCTL 1.2, BIMAS, SYFPEITHI and IEDB. The modified peptides from MUC4 containing HLA-A2 were obtained by replacing anchor residues of the binding anchor motifs. The peptides were synthesized by standard solid-phase methods. The binding affinity of the peptides to HLA-A2 molecule was evaluated by T2 binding assay. ELISPOT assay was used to investigate the ability of the peptide to induce specific restricted cytotoxic T-lymphocytes （CTLs） and release of IFN-γ. The ability of the peptides to induce T-cell response was investigated by cytotoxicity assay in vitro.RESULTS： The expression of MUC4 was observed in the CAPAN-2 cells and ASPC-1 cells. The candidate peptides P1944-1Y, P1944-2L, P1944-1Y2L, P2004 and P2004-1Y9V showed moderate affinity toward HLA-A2 molecule. T2 binding assay showed that P1944-1Y2L and P2004-1Y9V had significantly higher affinity for HLA-A2 than the native peptides. ELISPOT assay showed P1944, P1944-1Y2L, P2004 and P2004-1Y9V were able to induce specific CTLs and more amounts of IFN-γ were released. ELISPOT assay showed that significantly more amounts of IFN-γ released by P1944-1Y2L and P2004-1Y9V were observed than the native peptides. The CTLs induced by P1944, P1944-1Y2L, P2004 and P2004-1Y9V lyzed the CAPAN-2 cells. P1944-1Y2L and P2004-1Y9V peptide-specific CTLs showed higher cytotoxicity against pancreatic tumor cell line CAPAN-2 than the native peptide-specific CTLs.CONCLUSION： Compared with the native peptides, modified epitopes P1944-1Y2L and P2004-1Y9V have higher binding affinity with HLA-A2 and retain immunogenecity. In addition, the anti-tumor immunity of modified epitopes P1944-1Y2L and P2004-1Y9V is stronger than that of the native pept

关 键 词：黏蛋白4 表位 细胞毒性T淋巴细胞 

分 类 号：Q279[生物学—细胞生物学] R730.23[医药卫生—肿瘤]