新冠病毒受体蛋白ACE2结构与功能的生信分析及原核表达  

Bioinformatics analysis on structure and function of SARS-CoV-2 receptor protein ACE2 and its prokaryotic expression

作　　者：徐本锦 陈晓聪 宣焱 杜淼 范蕾 李卓禧 李璟 刘玲 宋彬妤[3] 侯艳香 XU Benjin;CHEN Xiaocong;XUAN Yan;DU Miao;FAN Lei;LI Zhuoxi;LI Jing;LIU Ling;SONG Binyu;HOU Yanxiang(Department of Medical Laboratory,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基础医学部,汾阳032200 [3]山西医科大学汾阳学院科技中心,汾阳032200

出　　处：《中国免疫学杂志》2023年第6期1230-1237,共8页Chinese Journal of Immunology

基　　金：山西省高等学校科技创新项目(2020L0749);山西医科大学汾阳学院引进人才启动金项目(2020A01);国家级大学生创新创业训练计划项目(202117114001);山西省高等学校大学生创新创业训练计划重点项目(S202117114008);山西省高等学校大学生创新创业训练计划重点项目(20221577);山西省基础研究计划(自由探索类)项目(20210302123397,202203021212351);吕梁市科技计划项目(2020SHFZ29)资助。

摘　　要：目的:构建ACE2原核表达载体并对其进行结构与功能的生物信息学分析,探究其介导SARS-CoV-2入侵人体的分子机制。方法:采用Protparam、ProtScale、NetPhos3.1、SignaIP 4.1、YinOYang 1.2 Server、NetNGlyc 1.0 Server、SOPMA、SWISS-MODEL、Blast、Clustal X2和MEGA7.0等对ACE2的生物学特性、同源性及进化性进行系统分析。采用分子克隆技术构建pET-22b-ACE2,在大肠杆菌中进行表达。结果:ACE2全长805个残基,呈酸性,分子量为92.5 kD,pI=5.36,共含77个潜在的磷酸化位点和14个糖基化位点,是一种亲水性较强的跨膜蛋白。ACE2主要散布于内质网膜、高尔基体和胞质膜,二级结构组成以α-螺旋为主。原核表达结果显示,细菌裂解后,沉淀中ACE2表达多于上清与全菌,为疫苗研发提供了理论依据。多序列比对和进化分析显示,倭黑猩猩与智人亲缘关系最近。结论:本研究为ACE2蛋白的纯化及研究奠定了基础,有助于阐明ACE2介导SARS-CoV-2入侵人体的分子机制,对SARS-CoV-2感染治疗和跨物种传播提供了新的见解,为研究广谱抗病毒药物、治疗SARS-CoV-2和其他致命冠状病毒株的有效分子靶点提供了依据。Objective:To construct prokaryotic expression vector of ACE2,to analyze its structure and function by bioinfor-matics and to explore molecular mechanism of SARS-CoV-2 invasion into human body.Methods:Biological characteristics,homology and evolution were systematically analyzed by Protparam,ProtScale,NetPhos3.1,SignaIP 4.1,YinOYang 1.2 Server,NetNGlyc 1.0 Server,SOPMA,SWISS-MODEL,Blast,Clustal X2 and MEGA7.0.pET-22b-ACE2 was constructed by molecular cloning technology and expressed in E.coli.Results:ACE2 was an acidic transmembrane protein with 805 amino acids,whose molecular weight was 92.5 kD,pI=5.36,had 77 potential phosphorylation sites and 14 glycosylation sites.ACE2 was mainly distributed in endoplasmic reticulum,golgi apparatus and cell membrane of host cells,whose secondary structure was mainly composed ofα-helix.Prokaryotic expression results showed that ACE2 was mainly expressed in precipitation after centrifugation of bacterial lysate,which laid a founda-tion for vaccine development.Multiple sequence alignment and evolutionary analysis showed that Pan paniscus had closest genetic rela-tionship with Homo sapiens.Conclusion:This study provides a basis for purification and research of ACE2 protein,and helps to clarify molecular mechanism of ACE2 mediated SARS-CoV-2 invasion into human body,provides new insights into treatment and cross spe-doicies transmission of SARS-CoV-2 infection,and provides a basis for study of broad-spectrum antiviral drugs and effective molecular targets for treatment of SARS-CoV-2 and other deadly coronavirus strains.

关 键 词：SARS-CoV-2 载体构建 ACE2蛋白 生信分析 原核表达 

分 类 号：Q812[生物学—生物工程] R392[医药卫生—免疫学]