伪狂犬病毒PRV-2022毒株gD蛋白突变与人Nectin-1结合力  

Study on the binding ability of gD protein mutation of PRV-2022 strain to human Nectin-1

作　　者：王新悦 王维禹[2] 梅国勇 韩俊[1] 陈操[1] Wang Xinyue;Wang Weiyu;Mei Guoyong;Han Jun;Chen Cao(National Key Laboratory of Intelligent Tracking and Forecasting for Infectious Diseases,NHC Key Laboratory of Medical Virology and Viral Diseases,National Institute for Viral Disease Control and Prevention,Chinese Center for Disease Control and Prevention,Beijing 102206,China;College of Life Science and Agriculture and Forestry,Qiqihar University,Qiqihar 161006,China)

机构地区：[1]中国疾病预防控制中心病毒病预防控制所、传染病溯源预警与智能决策全国重点实验室、国家卫生健康委医学病毒和病毒病重点实验室,北京102206 [2]齐齐哈尔大学生命科学与农林学院,齐齐哈尔161006

出　　处：《中华实验和临床病毒学杂志》2024年第4期395-401,共7页Chinese Journal of Experimental and Clinical Virology

基　　金：国家重点研发计划(2023YFC2306000,2023YFC2308201);传染病预防控制国家重点实验室青年基金(2021SKLID504);国家病原微生物资源库(NPRC-32)。

摘　　要：目的为探究PRV-2022毒株gD蛋白不同突变对与Nectin-1受体结合的影响。方法通过PCR、RT-qPCR和基因测序技术对PRV-2022毒株进行鉴定,利用生物信息学方法对PRV-2022毒株gD基因进行遗传进化分析。体外构建PRV-2022毒株gD蛋白胞外域第69位和第82位氨基酸突变的重组表达质粒并表达纯化,通过His-pull down和生物层干涉技术比较了重组gD蛋白不同突变与Nectin-1受体的结合能力。结果从伪狂犬病毒PRV-2022毒株中调取gD基因,通过遗传进化分析发现PRV-2022毒株与2011年之前分离的毒株分属于同一个分支,遗传距离较近。成功构建含有A69V和S82N氨基酸突变的gD蛋白胞外域表达质粒,表达纯化得到重组PRV gD胞外域蛋白,比较发现gD-69、gD-82、gD-2022、gD-Bartha蛋白均能与Nectin-1相互作用。相对于PRV经典疫苗株Bartha,gD蛋白第69位和第82位氨基酸双突变后与人Nectin-1亲和力最高,而无论单独任一突变位点都使gD蛋白与Nectin-1的亲和力降低。结论PRV的gD蛋白存在A69V和S82N突变时明显影响与人Nectin-1受体结合能力且同时突变时与人Nectin-1亲和力最高。ObjectiveTo investigate the impact of various mutations in the gD protein of PRV-2022 strain on its binding to the Nectin-1 receptor.MethodsWe employed PCR,RT-qPCR and gene sequencing techniques for identification of the PRV-2022 strain.Furthermore,bioinformatics method were utilized to analyze the genetic evolution of the gD gene in PRV-2022 strain.Recombinant expression plasmid containing mutations at amino acids positions 69 and 82 within the extracellular domain of gD protein from PRV-2022 strain was constructed and expressed in vitro.The binding ability between different mutant forms of recombinant gD protein and Nectin-1 receptor was compared using His-pull down and biolayer interference techniques.ResultsThe gD gene of the PRV-2022 strain was obtained,and genetic evolution analysis revealed that the PRV-2022 strain belonged to the same branch as strains isolated prior to 2011,with a close genetic distance.The expression plasmids for gD extracellular domain containing A69V and S82N amino acid mutations were successfully constructed,enabling the expression and purification of recombinant PRV gD extracellular domain protein.Interaction studies demonstrated that gD-69,gD-82,gD-2022,and gD-Bartha proteins interacted with human Nectin-1.Notably,compared to the classical PRV vaccine strain Bartha,double mutation of amino acids 69 and 82 in the gD protein exhibited the highest affinity to human Nectin-1 receptor,whereas individual mutations at either site decreased this affinity.ConclusionsIntroduction of A69V and S82N mutations in the gD protein significantly affected its binding ability to human Nectin-1 receptor.Simultaneous occurrence of A69V and S82N mutations resulted in the highest affinity towards human Nectin-1 receptor.

关 键 词：伪狂犬病 伪狂犬病毒 gD蛋白 Nectin-1受体 分子相互作用 

分 类 号：R512.99[医药卫生—内科学]