新型冠状病毒E蛋白B细胞表位的预测及鉴定  

Prediction and identification of B-cell epitopes of SARS-CoV-2 E protein

作　　者：张鹏飞 刘钧 邹紫阳 康喜龙[1,2,3] 宋丽 焦新安 孟闯[1,2,3] 潘志明 ZHANG Peng-fei;LIU Jun;ZOU Zi-yang;KANG Xi-long;SONG Li;JIAO Xin-an;MENG Chuang;PAN Zhi-ming(Jiangsu Provincial Key Laboratory of Zoonoses,Yangzhou University,Yangzhou 225009,China;Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses,Yangzhou University,Yangzhou 225009,China;Key Laboratory of Prevention and Control of Biological Hazard Factors(Animal Origin)for Agrifood Safety and Quality,Ministry of Agriculture and Rural Affairs,Yangzhou University,Yangzhou 225009,China)

机构地区：[1]扬州大学,江苏省人兽共患病学重点实验室,扬州225009 [2]扬州大学,江苏省动物重要疫病与人兽共患病防控协同创新中心,扬州225009 [3]扬州大学,农业农村部农产品质量安全生物性危害因子(动物源)控制重点实验室,扬州225009

出　　处：《中国人兽共患病学报》2024年第9期807-813,共7页Chinese Journal of Zoonoses

基　　金：国家重点研发计划项目(No.2022YFC2604200);江苏省重点研发计划项目(No.BE2021331);江苏省第六期“333高层次人才培养工程”项目联合资助。

摘　　要：目的利用生物信息学方法预测SARS-CoV-2 E蛋白B细胞表位,并利用小鼠多抗血清、人新型冠状病毒阳性血清等进行验证,以明确SARS-CoV-2 E蛋白的优势B细胞表位。方法使用SOPMA、Expasy、SWISS-MODEL及IEDB数据库和BepiPred-2.0等软件预测SARS-CoV-2 E蛋白的结构及B细胞表位;通过大肠杆菌系统表达并纯化GST标签重组表位蛋白片段,以Western blotting和间接ELISA方法检测表位蛋白与小鼠和人SARS-CoV-2 E蛋白阳性多抗血清的反应性,以初步鉴定SARS-CoV-2 E蛋白的B细胞表位。结果表位预测结果显示,E蛋白含有线性B细胞表位Ser6-Val14和Tyr57-Pro71,构象表位涉及的氨基酸序列为Glu8-Val14、Leu39-Tyr59、Ser60-Leu65;表达并纯化含有预测表位的E蛋白片段E1(Ser6-Val14表位)、E3(Tyr57-Pro71)以及阴性对照片段E2(不含表位序列),Western blotting和间接ELISA结果均显示抗E蛋白小鼠多抗和人新型冠状病毒阳性血清只与E1、E3蛋白片段呈阳性反应而与E2蛋白片段均为阴性反应,显示E蛋白线性B细胞表位预测正确。结论本研究成功预测并初步鉴定出SARS-CoV-2 E蛋白2个线性B细胞表位,为新型冠状病毒疫苗制备和免疫应答特性分析等提供参考。This work was aimed at predicting and verifying B-cell epitopes of SARS-CoV-2 E protein through bioinformatics methods,and clarifying the dominant B cell epitopes with mouse polyclonal antibody serum prepared through SARS-CoV-2 recombinant E protein immunization and human positive serum vaccinated with inactivated SARS-CoV-2 vaccine.The structural and B-cell linear epitopes of SARS-CoV-2 E protein were predic ted with SOPMA,Expasy,SWISS-MODEL,IEDB database,and Bepid-2.0 software.Candidate epitopes were expressed as GST-tagged recombinant protein fragments in an E.coli system,and their immunoreactivity with mouse and human polyclonal positive serum against SARS-CoV-2 E protein was detected by western blotting and indirect ELISA,respectively.The epitope prediction results showed that E protein contained linear B cell epitopes Ser6-Val14 and Tyr57-Pro71,and the conformational epitopes of Glu8-Val12,Leu39-Tyr59,and Ser60-Leu65.The GST tagged recombinant E protein fragments of E1 and E3,containing Ser6-Val14 and Tyr57-Pro71 epitopes,respectively,as well as E2 without an epitope sequence as a control,were expressed in an E.coli expression system and successfully purified with an Ni-NTA column.Western blotting and indirect ELISA analysis indicated that all mouse and human SARS-CoV-2 positive sera positively reacted with only E1 and E3 proteins,but negatively reacted with E2 protein,thus indicating that the corresponding epitope prediction with Ser6-Val14 and Tyr57-Pro71 was correct.This study successfully predicted and preliminarily identified two linear B cell epitopes of SARS-CoV-2 E protein,thus providing a reference for the preparation of new coronavirus vaccines and the analysis of immune response characteristics.

关 键 词：新型冠状病毒 B细胞表位 线性表位 预测 鉴定 

分 类 号：R373.1[医药卫生—病原生物学]