胰腺癌高表达抗原MUC4 CTL表位肽的筛选与改造

发布时间：2018-03-07 15:20

本文选题：黏蛋白　切入点：表位　出处：《中国病理生理杂志》2017年05期 　论文类型：期刊论文

【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。
[Abstract]:Objective: to observe whether the modified epitope of mucin 4) of pancreatic cancer has HLA-A2 restricted anti-tumor ability. Methods: firstly, the expression of MUC4 in the pancreatic cancer cell line CAPAN-2 and ASPC-1 was detected by RT-PCR and Western blot. The expression of MUC4 in the pancreatic cancer cell line CAPAN-2 and ASPC-1 was detected by Net. CTL 1.2 BIMASA SYFPEITHI and IEDB software were used to predict and score MUC4 HLA-A2 restricted epitopes. The modified peptide was obtained by replacing the amino acid at the anchoring site of MUC4 antigen, and the candidate epitope peptide was 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 cytotoxic T lymphocytes to secrete IFN- 纬. In vitro cytotoxicity assay activity was used to detect the ability of candidate peptides to induce CTL. Results the expression of 1: MUC4 in both pancreatic cancer cell lines CAPAN-2 and ASPC-1. P1944-1YP1944-2LP1944-1Y2LP2004 and P2004-1Y9V had good binding ability. The binding ability of modified peptide P1944-1Y2L to HLA-A2 was higher than that of propeptide. Elispot. The results showed that CTL induced by epitope peptide P1944 and P2004-1Y9V had the ability to secrete IFN- 纬. P1944-1Y2L and P2004-1Y9V induced specific T cell immune secretion, IFN- 纬 was slightly higher than that induced by P2004-1Y9V. The cytotoxicity of P1944-1Y2LP2004 and P2004-1Y9V on CAPAN-2 cells was higher than that on CAPAN-2 cells by P1944-1Y9V specific CTLs. Conclusion compared with P1944-1Y2L modified epitope P1944-1Y2LP2004-1Y9V, P444-1Y2LP141Y9V has higher affinity for HLA-A2 molecules than P1944-1Y9V, and P2004-1Y9V has higher affinity to CAPAN-2 cells than P1944-1Y9V. The original immunogenicity was retained, and the antitumor effect of modified peptide was stronger than that of natural epitope. P1944-1Y2L and P2004-1Y9V were excellent candidate epitopes of HLAA2 restricted CTL for MUC4 antigen, which could be used as candidate epitopes of new antitumor peptide immunotherapy vaccine.
【作者单位】：漯河医学高等专科学校医学生物工程重点实验室;
【基金】：河南省科技厅科技发展计划项目(No.142102310203)
【分类号】：Q279;;R730.23

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