新冠病毒核衣壳蛋白结构与功能的生物信息学分析及原核表达

Bioinformatics analysis for structure and function of nucleocapsid protein of SARS-CoV-2 and its prokaryotic expression

目的:对SARS-CoV-2核衣壳(N)蛋白进行系统生物信息学分析和原核表达,阐明其功能和病毒RNA复制机制。方法:采用UniProt、NetPhos 3.1、IEDB和Blast等生物信息学软件对N蛋白的理化性质、蛋白互作网络、翻译后修饰、同源性及系统进化关系等生物学特性进行系统分析。构建原核表达质粒pET-22b-N并表达蛋白。结果:N蛋白是由419个氨基酸组成的碱性蛋白,分子量为45.62 kD,等电点为10.07,其N端有1个RNA结合结构域,C端有1个二聚体界面,有57个可能的磷酸化修饰和25个潜在糖基化修饰,15个T细胞抗原表位和11个线性B细胞抗原表位,无信号肽和跨膜螺旋,是一种亲水性蛋白。N蛋白中无规则卷曲占比最高(54.42%),其次为α-螺旋(22.20%)。与N蛋白序列一致性最高的是BtRs-β-冠状病毒/HuB2013和蝙蝠冠状病毒Rp/Shaanxi2011,进化分析显示SARS-CoV-2与蝙蝠非典型冠状病毒YNLF_31C聚为一支。原核表达后发现,细菌裂解液离心后的沉淀中有大量N蛋白表达,为后续蛋白纯化和结构分析提供了依据。结论:N蛋白在SARS冠状病毒和蝙蝠冠状病毒间高度保守,为SARS-CoV-2 N蛋白结构与功能研究提供了基础,为靶向N蛋白的抗病毒药物设计提供了依据。

Objective:To expound function and replication mechanism of viral RNA of nucleocapsid(N)protein of SARSCoV-2 by systematic bioinformatics analysis and prokaryotic expression.Methods:Bioinformatics software such as UniProt,NetPhos 3.1,IEDB and Blast were used to systematically analyze physical and chemical properties,protein-protein interaction network,posttranslational modification and homology and phylogenetic relationship of N protein.Prokaryotic expression plasmid pET-22b-N was constructed and the protein was expressed.Results:N protein was a basic protein composed of 419 amino acids,with a molecular weight of 45.62 kD and an isoelectric point of 10.07,which has an RNA binding domain in N-terminal,a dimer interface in C-terminal,57 possible phosphorylation modification and 25 potential glycosylation modification,15 T-cell epitopes and 11 linear B-cell epitopes,was a hydrophilic protein without signal peptide and transmembrane helix.Irregular coil accounted for highest proportion of N protein(54.42%),followed byα-helix(22.20%).BtRs-β-CoV/HuB2013 and Bat coronavirus Rp/Shaanxi2011 had highest sequence identity with N protein.Phylogenetic analysis showed that SARS-CoV-2 and bat SARS-like coronavirus YNLF_31C clustered into one branch.After prokaryotic expression,it was found that N protein was mainly expressed in the precipitation after centrifugation of bacterial lysate,which laid a foundation for subsequent protein purification and structural analysis.Conclusion:N protein is highly conserved between SARS coronavirus and bat coronavirus,which provides an important basis for study of structure and function of SARS-CoV-2N protein,and provides a basis for design of antiviral drugs targeting N protein.