汉滩病毒糖蛋白Gn的T细胞表位预测及其跨种属验证  

Prediction of T cell epitopes of Hantavirus glycoprotein Gn and cross species validation

作　　者：孙报增 秦聪聪 张俊琦 王永凯 张文标 刘瑞波 白天原 张志辉 张宇丝 杨琨 姜东伯 Sun Bao-Zeng;Qin Cong-Cong;Zhang Jun-Qi;Wang Yong-Kai;Zhang Wen-Biao;Liu Rui-Bo;Bai Tian-Yuan;Zhang Zhi-Hui;Zhang Yu-Si;Yang Kun;Jiang Dong-Bo(Department of Immunology,Basic Medicine School,Air Force Medical University,Xi'an,Shaanxi 710032,China;Key Laboratory of Biological Weapon Damage and Prevention Drugs,Basic Medicine School,Air Force Medical University,Xi'an,Shaanxi 710032,China;Department of Microbiology,Basic Medicine School,Air Force Medical University,Xi'an,Shaanxi 710032,China;Department of Rheumatology,Tangdu Hospital,Air Force Medical University,Xi'an,Shaanxi 710032,China)

机构地区：[1]空军军医大学基础医学院免疫学教研室,陕西西安710032 [2]空军军医大学基础医学院生物武器损伤防治药物重点实验室,陕西西安710032 [3]空军医科大学唐都医院风湿科,陕西西安710032 [4]空军军医大学基础医学院微生物学教研室,陕西西安710032

出　　处：《解放军医学杂志》2023年第9期1000-1010,共11页Medical Journal of Chinese People's Liberation Army

基　　金：国家自然科学基金面上项目(82073154);国家自然科学基金青年项目(82203510);空军军医大学提升计划课题项目(2020XXXX03)。

摘　　要：目的筛选、鉴定并验证汉滩病毒(HTNV)包膜糖蛋白-N末端(Gn)上的免疫反应性表位。方法通过UniProt数据库获取HTNV 76-118株Gn蛋白序列。采用IEDB、SMMPMBEC、NetMHCpan 4.1、SYFPEITHI、Rankpep算法预测表位亲和力,VaxiJen v2.0分析免疫原性,Blastp工具计算保守性,HPEPDOCK和EpiDOCK模拟pMHC对接,TBtools进行双向聚类分析,酶联免疫斑点(ELISpot)实验评价表位细胞免疫反应性。结果通过UniProt数据库获得了HTNV 76-118株Gn蛋白序列(PRO_0000036816)。整合5种亲和力算法,在小鼠H-2亚型中获得了61个优势表位,在人主要组织相容性复合物(MHC)-Ⅰ亚型中得到234个优势表位,MHC-Ⅱ亚型中得到212个优势表位;VaxiJen筛选分别获得H-2、MHC-Ⅰ、MHC-Ⅱ亚型强免疫原性优势表位23、110、42个;进一步采用Blastp筛选获得高亲和力、强免疫原性、种间种内保守的MHC-Ⅰ类限制性表位3个,MHC-Ⅱ类限制性表位81个。双向分层聚类分析揭示了小鼠H2-d、H2-b和部分人类白细胞分化抗原(HLA)在提呈HTNV Gn表位上的相似性。ELISpot验证了5个表位具有较强的诱导脾细胞分泌γ干扰素(IFN-γ)的能力。结论预测并验证了HTNV Gn上的细胞免疫反应性表位,证实在MHC提呈中病毒抗原存在跨基因、种属、物种的交叉免疫反应性,为预防肾综合征出血热(HFRS)提供了新思路。Objective To screen,identify and validate the immunoreactive epitopes on the glycoprotein-N terminal(Gn)of Hantaan virus(HTNV)for providing a new idea for prevention of hemorrhagic fever with renal syndrome(HFRS).Methods The Gn protein sequences of HTNV strain 76-118 were obtained from UniProt database.IEDB,SMMPMBEC,NetMHCpan 4.1,SYFPEITHI and Rankpep were used to predict epitope affinity.Immunogenicity was analyzed by VaxiJen.Blastp analyzed the conservation;HPEPDOCK and EpiDOCK simulated pMHC docking;TBtools implemented bidirectional cluster analysis;Immunoreactivity of epitope in vivo was evaluated by enzyme-linked immunospot assay.Results The Gn protein sequence of HTNV 76-118 strain(PRO_0000036816)was obtained from UniProt database.Five affinity algorithms were integrated to obtain 61 dominant epitopes in mouse H-2 subtype,234 dominant epitopes in human major histocompatibility complex(MHC)-Ⅰsubtype,and 212 dominant epitopes in MHC-Ⅱsubtype.VaxiJen screening obtained 23,110 and 42 dominant epitopes of H-2,MHC-Ⅰand MHC-Ⅱsubtypes,respectively.Further Blastp screening resulted in 3 MHC-Ⅰrestricted epitopes with high affinity and strong immunogenicity,and 81 MHC-Ⅱrestricted epitopes conserved between species.Bidirectional hierarchical cluster analysis revealed the similarity of HTNV Gn epitopes in H2-d,H2-b of mice and some human leucocyte antigen(HLA).ELISpot verified that 5 epitopes could induce splenic cells to secrete interferon-gamma(IFN-γ).Conclusions The present study predicted and verified the cellular immunoreactive epitopes on HTNV Gn that can induce cellular immune reactivity,revealed the cross activity across-genes,species and genera immunoreactivity of viral antigens in MHC presentation,and provide guidance for the development of novel HFRS epitope vaccines.

关 键 词：汉滩病毒 糖蛋白 表位 计算机预测 免疫反应性 

分 类 号：R512.8[医药卫生—内科学] R373.3[医药卫生—临床医学]