本文選題：黏蛋白　切入點(diǎn)：表位　出處：《中國病理生理雜志》2017年05期 　論文類型：期刊論文

【摘要】：目的:觀察胰腺癌高表達(dá)抗原黏蛋白4(MUC4)的改造表位是否有HLA-A2限制性抗腫瘤能力。方法:首先運(yùn)用RT-PCR和Western blot方法檢測MUC4在胰腺癌細(xì)胞系CAPAN-2和ASPC-1的表達(dá)情況。通過Net CTL 1.2、BIMAS、SYFPEITHI和IEDB軟件預(yù)測打分來選取MUC4的HLA-A2限制性表位;替換MUC4抗原錨定位點(diǎn)氨基酸獲得改造肽;候選表位肽的合成方法為標(biāo)準(zhǔn)的Fmoc化學(xué)合成法,結(jié)合力實(shí)驗(yàn)用于檢測候選表位與T2A2細(xì)胞表面HLA-A2分子的結(jié)合能力,ELISPOT實(shí)驗(yàn)檢測候選表位肽誘導(dǎo)細(xì)胞毒性T淋巴細(xì)胞(CTL)分泌IFN-γ的能力,體外細(xì)胞毒實(shí)驗(yàn)活性檢測候選肽誘導(dǎo)CTL的能力。結(jié)果:MUC4在胰腺癌細(xì)胞系CAPAN-2和ASPC-1均有表達(dá)。P1944-1Y、P1944-2L、P1944-1Y2L、P2004和P2004-1Y9V具有較好的結(jié)合力,且P2004-1Y9V、P1944-1Y2L等改造肽與HLA-A2的結(jié)合力高于原肽。ELISPOT實(shí)驗(yàn)結(jié)果顯示表位肽P1944、P1944-1Y2L、P2004和P2004-1Y9V誘導(dǎo)的CTL具有分泌IFN-γ的能力。P1944-1Y2L和P2004-1Y9V誘導(dǎo)特異性T細(xì)胞免疫分泌的IFN-γ略高于原肽。細(xì)胞毒實(shí)驗(yàn)結(jié)果顯示表位P1944、P1944-1Y2L、P2004和P2004-1Y9V對CAPAN-2細(xì)胞均有一定的殺傷作用。P1944-1Y2L和P2004-1Y9V特異性CTLs對CAPAN-2細(xì)胞殺傷率高于原肽特異性CTLs。結(jié)論:MUC4抗原改造表位P1944-1Y2L、P2004-1Y9V與天然表位P1944、P2004相比有更高的HLA-A2分子親和力,保留了原有的免疫原性,并且改造肽抗腫瘤免疫效應(yīng)強(qiáng)于天然表位。P1944-1Y2L和P2004-1Y9V是優(yōu)秀的MUC4抗原的HLAA2限制性CTL候選表位,可以成為新的抗腫瘤多肽免疫治療疫苗的候選表位。
[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- 緯. 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- 緯. P1944-1Y2L and P2004-1Y9V induced specific T cell immune secretion, IFN- 緯 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.
【作者單位】： 漯河醫(yī)學(xué)高等�？茖W(xué)校醫(yī)學(xué)生物工程重點(diǎn)實(shí)驗(yàn)室;
【基金】：河南省科技廳科技發(fā)展計(jì)劃項(xiàng)目(No.142102310203)
【分類號】：Q279;;R730.23

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