基于骨肉瘤高表达抗原PBF CTL表位改造及鉴定

发布时间：2018-07-27 19:23

【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。
[Abstract]:Aim: to investigate whether the modified epitope of high expression antigen papillomavirus binding factor (PBF) of osteosarcoma has HLA-A2 restricted antitumor ability and to develop polypeptide immunotherapy based on osteosarcoma. Methods: RT-PCR and Western blot were used to detect the expression of PBF in osteosarcoma cell line U2OS and Saos-2. Then the HLA-A2 restricted epitopes of PBF were selected by Net CTL 1.2 Syssypetra and IEDB software prediction scoring. The modified peptide was obtained by replacing the amino acid at the anchoring site of PBF antigen. Candidate epitope peptides are synthesized by the standard Fmoc chemical synthesis method. The binding ability of candidate epitopes to HLA-A2 molecules on the surface of T2A2 cells was determined by binding assay. Elispot assay was used to detect the ability of candidate epitope peptides to induce the secretion of interferon 纬 (IFN- 纬) by (CTL) from cytotoxic T lymphocytes. The ability of candidate peptides to induce CTL was detected by lactate deoxylase (LDH) release assay and carboxyl fluorescein succinimide (CFSE) cytotoxicity assay. Results both U2OS and Saos-2 were expressed in human osteosarcoma cell line. The candidate peptide P75-1Y2LP412-1YP416-1Y2L9VX P107-1Y and P435-1Y2L had good binding ability. The binding ability of modified peptide to HLA-A2 was higher than that of propeptide. Elispot. The results showed that the epitope peptide P412P412-1YP416P416-1Y2L9V and CTL induced by P435-1Y2L had the ability to secrete IFN- 纬. P412-1Y and P416-1Y2L9V induced specific T cell immune secretion were slightly higher than those of propeptide. LDH release test and CFSE cells. The results showed that the cytotoxicity of epitope P412-1YP416 and P416-1Y2L9V to U2OS cells was higher than that of P412-1Y and P416-1Y2L9V specific CTLs. Conclusion compared with the natural epitopes P412 and P416, the modified epitopes P412-1Y and P416-1Y2L9V have higher HLA-A2 molecular affinity and retain the original immunogenicity. The modified peptides have stronger antitumor immune effect than natural epitopes. P412-1Y and P416-1Y2L9V are candidate epitopes of HLA-A*0201 restricted CTL for excellent PBF antigens, which can be used as candidate epitopes for new antitumor polypeptide immunotherapy vaccines.
【作者单位】：许昌学院;漯河医学高等专科学校;
【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)
【分类号】：R738.1

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