新冠病毒刺突糖蛋白结构与功能的生物信息学分析及原核表达  

Bioinformatics Analysis on Structure and Function of the Spike Glycoprotein of SARS-CoV-2 and Its Prokaryotic Expression

作　　者：徐本锦 宣焱 杜淼 刘玲 吴惠文[2] 宋彬妤[2] 侯艳香 陈利荣 刘晓梁[2] XU Benjin;XUAN Yan;DU Miao;LIU Ling;WU Huiwen;SONG Binyu;HOU Yanxiang;CHEN Lirong;LIU Xiaoliang(Department of Medical Laboratory,Fenyang College of Shanxi Medical University,Fenyang 032200,China;Science and Technology Centre,Fenyang College of Shanxi Medical University,Fenyang 032200,China)

机构地区：[1]山西医科大学汾阳学院医学检验系,汾阳032200 [2]山西医科大学汾阳学院科技中心,汾阳032200

出　　处：《病毒学报》2022年第1期41-56,共16页Chinese Journal of Virology

基　　金：山西医科大学汾阳学院引进人才启动金项目(项目号:2020A01),题目:RelA介导大肠杆菌严谨反应的分子机制研究;山西省高等学校科技创新项目(项目号:2020L0749),题目:大肠杆菌核糖体再循环因子RRF的功能研究;国家级大学生创新创业训练计划项目(项目号:202117114001),题目:新冠病毒Nsp1基因的克隆、表达载体构建及其对大肠杆菌生长的影响;山西省高等学校大学生创新创业训练计划重点项目(项目号:S202117114008),题目:SARS-CoV-2核衣壳蛋白过表达影响细菌蛋白翻译的分子机制;吕梁市科技计划项目(项目号:2020SHFZ29),题目:吕梁市食源性“超级耐药菌”MRSA的遗传多态性与溯源研究;吕梁市科技计划项目(项目号:2020XGZX104),题目:新冠肺炎疾病转归预测和风险评估的模型构建与评估。

摘　　要：SARS-CoV-2是一种高致病性且传播迅速的病原体,通过刺突糖蛋白(Spike glycoprotein,S蛋白)识别宿主细胞表面的受体来实现入侵和感染。对S蛋白进行系统的生物信息学分析和原核表达,有助于深入理解S蛋白的功能和阐明该蛋白介导病毒感染的分子机制。本文采用Protparam、Pfam、TMHMM、ExPASy-ProtScale、PSORTⅡ、SignalP、UniProt、NetPhos 3.1、NetNGlyc 1.0、NetOGlyc 4.0和BLAST等生物信息学软件和数据库对S蛋白的理化性质、亚细胞定位、翻译后修饰及相互作用网络等生物学特性进行了系统分析。利用Clustal X2和MEGA7.0软件对该蛋白进行了基于氨基酸序列的同源性分析和系统进化分析。最后,通过分子克隆技术构建重组表达载体pET-22b-S并进行原核表达。结果显示,S蛋白由1273个氨基酸组成,分子量141.2 kD,等电点6.24,有两个卷曲螺旋结构,一个跨膜螺旋区,疏水性较强。S蛋白包含刺突受体结合结构域和S2糖蛋白结构域,主要分布于宿主细胞的内质网膜和细胞膜,含有136个潜在的磷酸化位点和20个可能的糖基化位点。与SARS-CoV-2 S蛋白序列一致性最高的是SARS冠状病毒、SARS冠状病毒WH20和蝙蝠冠状病毒HKU3,均为76%。SARS-CoV-2与SARS冠状病毒和蝙蝠冠状病毒聚为一大支,提示它们可能具有共同的祖先。S蛋白主要在细菌裂解液离心之后的沉淀中表达,这为后续的结构分析和疫苗研发奠定了基础。S蛋白在SARS冠状病毒和蝙蝠冠状病毒之间保守性较高,提示其在病毒入侵过程中具有重要功能。SARS-CoV-2与SARS冠状病毒和蝙蝠冠状病毒可能具有共同的祖先。本研究为SARS-CoV-2 S蛋白的表达纯化、结构与功能研究提供了重要的数据基础,有助于全面揭示S蛋白的生物学功能,同时为设计和筛选靶向S蛋白的新型抗病毒药物提供了科学依据。Severe acute respiratory syndrome-coronavirus 2(SARS-CoV-2)is a highly pathogenic and rapidly spreading pathogen.It can invade and infect cells by recognizing receptors on the surface of host cells with spike(S)glycoprotein.Systematic bioinformatics analysis and prokaryotic expression of the S protein can aid understanding of its function and clarify the molecular mechanism of viral infection mediated by this protein.The physicochemical properties,subcellular localization,post-translational modifications and protein-interaction network of the S protein were analyzed systematically using Protparam,Pfam,TMHMM,ExPASy-ProtScale,PSORTⅡ,SignalP,UniProt,NetPhos 3.1,NetNGlyc 1.0,NetOGlyc 4.0,BLAST and other bioinformatics software and databases.In addition,Clustal X2 and MEGA7.0 were used to analyze the homology and phylogeny of S glycoproteins based on amino-acid sequences.Finally,recombinant expression vector of the S glycoprotein was constructed by molecular cloning technology and expressed in Escherichia coli.Results showed that the S glycoprotein is composed of 1,273 amino acids,with a molecular weight of 141.2 kD and an isoelectric point of 6.24.It had two coiled helical structures and one transmembrane helix region.It was a hydrophobic protein,contained a spike receptor-binding domain and S2 glycoprotein domain.This protein was distributed mainly in the endoplasmic-reticulum membrane(39.1%)and cell membrane(21.7%)of host cells,and contained 136 potential phosphorylation sites and 20 possible glycosylation sites.SARS-CoV,SARS-CoV WH20 and bat coronavirus HKU3 showed the highest sequence identity with the spike-glycoprotein sequence of SARS-CoV-2(76%).SARS-CoV-2,SARS-CoV and bat coronavirus clustered into a large branch,suggesting that they may have a common ancestor.The S protein was expressed mainly in the precipitate after centrifugation of bacterial lysates,which lays a foundation for future structural analysis and vaccine development.The S glycoprotein was highly conserved between SARS-CoV and bat coronavirus

关 键 词：新冠病毒(SARS-CoV-2) 刺突糖蛋白(S蛋白) 生物信息学分析 原核表达 

分 类 号：Q71[生物学—分子生物学]