人端粒酶逆转录酶抗原HLA-A0201限制性CTL表位预测及鉴定

Prediction and identification of HLA-A0201 restricted CTL epitopes derived from human telomerase reverse transcriptase antigen

目的应用生物信息学方法对人端粒酶逆转录酶(h TERT)HLA-A2+限制性细胞毒性T细胞(CTL)表位进行预测和鉴定,寻找诱导机体特异性杀伤肺癌肿瘤细胞的抗原表位。方法应用生物信息学软件BIMAS、SYFPEITHI对h TERT蛋白进行HLA-A0201限制性CTL抗原表位预测,筛选优势表位;应用肽亲和力实验、乳酸脱氢酶(LDH)释放实验及人干扰素γ(IFN-γ)ELISPOT实验验证表位,筛选出激发机体产生特异性免疫反应的表位。结果生物信息学软件筛选出优势表位为:ILAKFLHWL、ELLRSFFYV及ILSTLLCSL;肽亲和力实验得到优势表位荧光系数(FI)为:ILAKFLHWL0.67、ELLRSFFYV0.66及ILSTLLCSL0.90;LDH释放实验显示ILAKFLHWL所诱导CTLs的杀伤率明显高于其它各表位,也明显高于阴性表位,差异均具有统计学意义(P<0.05);人IFN-γELISPOT实验证明ILAKFLHWL所诱导的CTLs产生的IFN-γ斑点数多于其他表位,差异具有统计学意义(P<0.05)。结论ILAKFLHWL的免疫原性强,可用于后续制备肺癌多肽疫苗。

Objective HLA-A0201 restricted epitopes were screened for the hTERT protein, and the dominant epitopes were obtained. Then the immunogenicity of the dominant epitopes were detected, finally 1-2 stronger epitopes were acquired which could induced stronger immune response. Method applied bioinformaties software BIMAS and SYFPEITHI to predict hTERT protein HLA-A0201 restricted CTL epitopes. Then screened dominant epitopes according to the score of two kinds of software. Next, applied peptide affinity experiment, lactate dehydrogenase（LDH） release assay as well as human interfron gamma（IFN-γ） ELISPOT epitope screening experiments to veriify dominant epitopes. Finally, acquired the epitopes which had ability to elicit stronger immune respons. Results Comprehensive analysis of SYFPEITHI software and BIMAS software for HLA-A0201 restricted CTL antigen epitope prediction of hTERT protein showed that 3 peptides were better than others. They were ILAKFLHWL, ELLRSFFYV and ILSTLLCSL. The result of peptide affinty experiment showed that the fluorescence index （FI） of each peptide was ILAKFLHWL 0.67, ELLRSFFYV 0.66 and ILSTLLCSL 0. 90; The result of LDH releasing assay showed that the killing rate of ILAKFLHWL was significantly higher than other peptides and negative peptide（P〈0.05） ; The result of human IFN-γ ELISPOT test showed that the number of spots elicited by ILAKFLHWL epitope was significantly higher than other epitopes（P〈0.05）. Conclusions The PBMCs cells induced by epitope ILAKFLHWL had a most significant killing effect. The epitope ILAKFLHWL could be used to make lung cancer poly-peptide vaccine.