HLA-A0201、A1101和A2402限制性HBV抗原肽的虚拟预测与实验鉴定-东南大学学报医学版

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

作者：赵晨¹ 金萧萧¹ 丁艳¹ 昂倩倩¹ Odontuya Khaidav¹ 夏玲芝² 邱洁³ 沈传来¹

单位：1. 东南大学医学院病原生物学与免疫学系, 江苏南京 210009;
2. 南京金域医学检验所自身免疫病检测中心, 江苏南京 210032;
3. 南京市第二医院传染病科, 江苏南京 210003

分类号：R373.21;R392.1

出版年·卷·期（页码）：2019·38·第六期（989-996）

摘要：

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-γ ELISPOT）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-γ ELISPOT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-γ enzyme 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.

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