基于骨肉瘤高表达抗原PBF CTL表位改造及鉴定  

Identification and molecular modification of cytotoxic T-lymphocyte epitopes from osteosarcoma high-expressing antigen PBF

作　　者：张建新[1] 时冉冉[2] 

机构地区：[1]许昌学院,河南许昌461000 [2]漯河医学高等专科学校,河南漯河462002

出　　处：《中国病理生理杂志》2017年第11期1993-1999,共7页Chinese Journal of Pathophysiology

基　　金：河南省科技厅科技发展计划项目(No.142102310203);漯河医学高等专科学校科研基金资助项目(No.2017-SLMC-3)

摘　　要：目的:观察骨肉瘤高表达抗原乳头瘤病毒结合因子(PBF)的改造表位是否有HLA-A2限制性抗肿瘤能力,开发基于骨肉瘤的多肽免疫治疗。方法:首先运用RT-PCR和Western blot方法检测PBF在骨肉瘤细胞系U2OS和Saos-2的表达情况。然后通过Net CTL 1.2、SYFPEITHI和IEDB软件预测打分来选取PBF的HLA-A2限制性表位。替换PBF抗原锚定位点氨基酸获得改造肽。候选表位肽的合成方法是标准的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)分泌干扰素γ(IFN-γ)的能力,乳酸脱氧酶(LDH)释放实验和羧基荧光素琥珀酰亚胺脂(CFSE)细胞毒实验检测候选肽诱导CTL的能力。结果:PBF在骨肉瘤细胞系U2OS和Saos-2均有表达,候选肽P75-1Y2L、P412-1Y、P416-1Y2L9V、P107-1Y和P435-1Y2L具有较好的结合力,且改造肽与HLA-A2的结合力高于原肽。ELISPOT实验结果显示表位肽P412、P412-1Y、P416、P416-1Y2L9V和P435-1Y2L诱导的CTL具有分泌IFN-γ的能力;P412-1Y和P416-1Y2L9V诱导特异性T细胞免疫分泌的IFN-γ略高于原肽。LDH释放实验和CFSE细胞毒实验结果显示表位P412、P412-1Y、P416和P416-1Y2L9V对U2OS细胞均有一定的杀伤作用,P412-1Y和P416-1Y2L9V特异性CTLs对U2OS细胞杀伤率高于原肽特异性CTLs。结论:PBF抗原改造表位P412-1Y和P416-1Y2L9V与天然表位P412和P416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。P412-1Y和P416-1Y2L9V是优秀的PBF抗原的HLA-A*0201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 osteosarcoma high-expressing antigen papillomavirus-binding factor（PBF） have HLA-A2 restricted antitumor ability,and to develop peptide-based immunotherapy for osteosarcoma.METHODS：RT-PCR and Western blot were used to determine the expression of PBF in the osteosarcoma cell lines U2 OS and Saos-2.HLA-A2 epitopes from PBF protein were predicted by Net CTL 1.2,SYFPEITHI and IEDB.The modified peptides from PBF containing HLA-A2 binding anchor motifs were designed by replacing the anchor residues.The peptides were synthesized by standard solid-phase methods,and the binding affinity of the peptides to HLA-A＊ 0201 was evaluated by T2 A2 cell binding assay.ELISPOT assay was used to investigate the seretion of interferon-γ（IFN-γ） from the peptide-induced specific cytotoxic T-lymphocytes（CTLs）.The ability of inducing T-cell response was analyzed by lactate dehydrogenase（LDH） release assay and carboxyfluorescein succinimidyl ester（CFSE） cytotoxicity assay in vitro.RESULTS：The expression of PBF was observed in the U2 OS and Saos-2 cells.The candidate peptides P75-1 Y2 L,P412-1 Y,P416-1 Y2 L9 V,P107-1 Y and P435-1 Y2 L showed moderate affinity toward HLA-A2 molecule.The modified peptides showed significantly higher affinity with HLA-A2 than the native peptide.ELISPOT assay showed that P412,P412-1 Y,P416,P416-1 Y2 L9 V and P435-1 Y2 L induced specific CTLs to secrete IFN-γ,and P412-1 Y and P416-1 Y2 L9 V induced more secretion of IFN-γ than the native peptide.The CTLs induced by P412,P412-1 Y,P416 and P416-1 Y2 L9 V lysed U2 OS cells.P412-1 Y and P416-1 Y2 L9 V peptide-specific CTLs showed higher cytotoxicity against U2 OS cells than the native peptide-specific CTLs.CONCLUSION：Compared with the native peptide,modified epitopes P412-1 Y and P416-1 Y2 L9 V have higher binding affinity with HLA-A＊ 0201 and retain immunogenecity.In addition,the anti-tumor immunity effects of modified epitopes P412-1 Y and P416-1 Y2 L9 V are stronger than the native

关 键 词：乳头瘤病毒结合因子 表位 细胞毒性T淋巴细胞 骨肉瘤 

分 类 号：R738.1[医药卫生—肿瘤] R392.3[医药卫生—临床医学]