基于生物信息学诺如病毒多表位抗原的构建及鉴定  

Construction and identification of multiple epitope antigens of norovirus based on bioinformatics

作　　者：杜雪 赵印震 张怡青 王晓军 王旭东 郭兰英 王云龙[3,5] DU Xue;ZHAO Yinzhen;ZHANG Yiqing;WANG Xiaojun;WANG Xudong;GUO Lanying;WANG Yunlong(Xinxiang Medical University,Xinxiang 453000,China;Luoyang Vocational&Technical College,Luoyang 471000,China;Zhengzhou Technical College,Zhengzhou 450000,China;Henan General Hospital,Zhengzhou 450000,China;Henan Bioengineering Technology Research Center,Zhengzhou 450000,China)

机构地区：[1]新乡医学院,新乡453000 [2]洛阳职业技术学院,洛阳471000 [3]郑州职业技术学院,郑州450000 [4]河南省职工医院,郑州450000 [5]河南省生物工程技术研究中心,郑州450000

出　　处：《中国免疫学杂志》2024年第11期2391-2398,共8页Chinese Journal of Immunology

基　　金：河南省中原学者工作站项目(224400510017)。

摘　　要：目的:基于生物信息学技术设计诺如病毒(NoV)的多表位抗原并进行制备及鉴定。方法:采用生物信息学方法构建NoV多表位抗原NoV-ZH并进行分析。通过原核表达系统制备重组蛋白并鉴定,经动物免疫、杂交瘤技术制备单克隆抗体,并在胶体金平台初步应用。结果:设计的多表位抗原二级结构中无规卷曲占比较大,理论分子质量及等电点(PI)为13.1 ku、7.16,性质稳定,亲水性较好。免疫评估显示具有良好的免疫原性,可激活体液免疫应答和细胞免疫应答。抗原序列连接pET-28a(+)、pET-32a载体后制备的蛋白分别以包涵体蛋白、可溶性蛋白的形式表达。经动物免疫、杂交瘤技术获得一对配对抗体,应用于胶体金试纸条灵敏度达0.5 ng/ml,试纸条可特异性结合两种基因型NoV重组衣壳蛋白。结论:成功制备诺如病毒多表位抗原并鉴定,为后续NoV通用型检测靶点探究及诊断原材料开发提供参考。Objective:To design a multi-epitope antigen of norovirus(NoV)based on bioinformatics technology and to pre-pare and characterize it.Methods:Bioinformatics methods were used to construct and analyze the NoV multi-epitope antigen NoV-ZH.Recombinant proteins were prepared and characterized by prokaryotic expression system,and monoclonal antibodies were prepared by animal immunization and hybridoma technology,and initially applied in colloidal gold platform.Results:The designed multi-epitope antigen had a large proportion of random curls in the secondary structure,with theoretical molecular mass and isoelectric point(PI)of 13.1 ku and 7.16,which were stable and hydrophilic.It had good immunogenicity and could activate humoral and cellular immune re-sponses.The proteins prepared by ligating pET-28a(+)and pET-32a vectors with antigenic sequences were expressed as inclusion body proteins and soluble proteins,respectively.A pair of paired antibodies was obtained by animal immunization and hybridoma tech-nique,and applied to colloidal gold test strips with a sensitivity of 0.5 ng/ml,and the test strips could specifically bind two genotypes of NoV recombinant capsid proteins.Conclusion:The successful preparation and characterization of multi-epitope antigen of norovirus provides a reference for the subsequent exploration of NoV universal detection targets and the development of diagnostic raw materials.

关 键 词：诺如病毒 生物信息学分析 多表位抗原 

分 类 号：Q819[生物学—生物工程]