HLA- A0201、A1101和A2402限制性HBV抗原肽的虚拟预测与实验鉴定

Virtual prediction and experimental identification of HBV antigenic peptides restricted by HLA- A0201, A1101 and A2402

目的:筛选和鉴定基因总频率>50%的3种人类白细胞抗原(HLA)(A0201、A1101和A2402)分子限制的乙型肝炎病毒(HBV)抗原T细胞表位肽。方法:利用6种在线T细胞表位肽预测数据库,针对乙肝病毒表面抗原、核心抗原、DNA多聚酶和X蛋白等4种抗原,虚拟筛选3种HLA-A分子限制的T细胞表位肽。从南京市第二医院检验科收集乙型肝炎住院患者的外周抗凝血,制备外周血单个核细胞,通过γ干扰素酶联免疫斑点法(IFN-γELISPOT)筛选出对任何一组混合多肽呈现特异T细胞反应的乙型肝炎患者;重新采集这些乙型肝炎患者的新鲜外周血,用IFN-γELISPOT法鉴定混合肽中单种抗原肽的免疫原性;并采用聚合酶链反应测序分型法对这些乙型肝炎患者进行HLA-A等位基因分型。结果:经鉴定具有免疫原性的HLA-A0201分子限制性HBV表位肽8种,A1101限制性HBV表位肽6种,A2402限制性HBV表位肽7种。结论:本研究筛选鉴定了3种HLA-A分子限制的4种HBV抗原的21种T细胞表位肽,其中11种未见报道,而且HLA-A1101分子限制性表位肽的免疫原性明显弱于HLA-A0201和A2402分子限制性表位肽。

Objective:To screen and identify the hepatitis B virus(HBV)antigenic T cell epitopes restricted by human leukocyte antigen(HLA)-A0201,A1101 and A2402 which have a total gene frequency of>50%in Chinese population.Methods:Six online data banks for T cell epitopes prediction were used to virtually screen the T cell epitopes derived from HBsAg,HBcAg,HBpol and HBx proteins and restricted by the three HLA-A molecules,respectively.Peripheral anticoagulation samples from HBV-infected in-patients were collected from the Department of Clinical Laboratory of Nanjing the Second Hospital,and peripheral blood mononuclear cells(PBMCs)were processed.Then the interferon-γenzyme linked immunospot(IFN-γ-ELISPOT)assay was performed to screen out the patients who had the specific T cell responses to the cocktail of peptides.The fresh peripheral blood samples were collected again from these patients and the immunogenicity of each peptide was confirmed by the IFN-γ-ELISPOT assay.Meanwhile,HLA-A genotyping was carried out in these HBV-infected patients through polymerase chain reaction-sequencing-based typing(PCR-SBT)method.Results:The immunogenicity of eight epitopes restricted by HLA-A0201,six epitopes restricted by A1101,and seven epitopes restricted by A2402 were confirmed by functional experiment.Conclusion:Twenty-one T cell epitopes have been identified,which derived from four kinds of HBV proteins and restricted by three HLA-A molecules,respectively.Of them,eleven epitopes have not been reported before.Moreover,the immunogenicity of the epitopes restricted by HLA-A1101 is significantly weaker than that restricted by HLA-A0201 and A2402.