基于酵母双杂交技术对与纳尔逊海湾正呼肠孤病毒σNS相互作用宿主蛋白的筛选  

Screening of host proteins interacting with Nelson Bay orthoreovirusσNS based on yeast two-hybrid technology

作　　者：孙绿茵 马竹萍 李润林 李永刚 陶晓莉 SUN Lyuyin;MA Zhuping;LI Runlin;LI Yonggang;TAO Xiaoli(Department of Pathogenic Biology,School of Basic Medical Sciences,Jinzhou Medical University,Jinzhou 121000,China)

机构地区：[1]锦州医科大学基础医学院病原生物学教研室,辽宁锦州121000

出　　处：《吉林大学学报（医学版）》2024年第5期1313-1321,共9页Journal of Jilin University：Medicine Edition

基　　金：辽宁省科技厅自然科学基金项目(2022-BS-320)。

摘　　要：目的:探讨小鼠成纤维L929细胞中与纳尔逊海湾正呼肠孤病毒(NBV)σNS蛋白相互作用(互作)的宿主蛋白,阐明宿主蛋白对病毒复制的影响。方法:构建表达σNS蛋白的诱饵质粒pGBKT7-S3,采用测序技术验证诱饵质粒在Y2H酵母细胞中的准确表达。将pGBKT7-S3和pGADT7质粒分别和共同转化至Y2HGold酵母细胞中,涂布于固体培养基进行培养,观察菌落生长情况,确认σNS蛋白对酵母细胞无毒性,不能自行激活报告基因。诱饵质粒pGBKT7-S3与小鼠成纤维L929细胞的cDNA文库进行杂交,对编码互作蛋白的阳性克隆进行质粒抽提,阳性测序结果通过Uniprot数据库检索筛选得到与NBVσNS互作的蛋白。对互作蛋白进行基因本体论(GO)功能富集分析、京都基因和基因组百科全书(KEGG)信号通路富集分析和STRING生物信息学分析。结果:成功筛选出61个阳性克隆。DNA测序分析和BLAST比对分析去除23个未匹配到数据库和测序基本相同的阳性克隆。阳性测序结果通过Uniprot数据库检索筛选得到38个与NBVσNS互作的蛋白,31个蛋白参与细胞生物过程,36个蛋白是细胞解剖成分,31个蛋白具有结合功能,5个蛋白是线粒体呼吸链组成部分,7个蛋白是核糖体蛋白和核糖体大小亚基的组成部分,2个蛋白参与铁代谢稳态。GO功能富集分析,互作蛋白参与的生物过程(BP)富集在细胞过程、代谢过程、生物学调节、定位和对刺激的反应等;细胞组分主要是细胞解剖成分和含蛋白复合物;分子功能集中在结合、催化活性、结构分子活性和转运活性等方面。KEGG信号通路富集分析,蛋白在遗传信息处理途径的翻译、折叠、分类和降解信号通路中高度富集,在机体系统中主要与消化系统有关联;与多种病毒性传染病和癌症有关联。STRING分析,互作蛋白涉及核糖体蛋白类、蛋白修饰类、代谢类和免疫蛋白类等功能蛋白。结结论论:成功筛选出与NBVσNS蛋�Objective:To discuss the host proteins that interact with the Nelson Bay orthoreovirus(NBV)σNS protein in the fibroblasts L929 of the mice,and to clarify the effect of host proteins on the viral replication.Methods:The bait plasmid pGBKT7-S3 expressingσNS protein was constructed,and sequencing technology was used to verify the accurate expression of the bait plasmid in the Y2H yeast cells.The pGBKT7-S3 and pGADT7 plasmids were separately and jointly transformed into the Y2HGold yeast cells,plated on solid medium,and the colony growth was observed to confirm that theσNS protein was non-toxic to the yeast cells and could not self-activate the reporter gene.The bait plasmid pGBKT7-S3 was hybridized with the cDNA library in fibroblasts L929 of the mice,and the plasmids encoding the interacting proteins were extracted from the positive clones.The positive sequencing results were screened for the proteins interacting with NBVσNS through the Uniprot database.Gene Ontology(GO)functional enrichment analysis,Kyoto Encyclopedia of Genes and Genomes(KEGG)signaling pathway enrichment analysis,and STRING bioinformatics analysis were performed on the interacting proteins.Results:A total of 61 positive clones were successfully screened.The DNA sequencing analysis and BLAST alignment removed 23 positive clones that did not match the database or were similar in sequence.The positive sequencing results identified 38 proteins interacting with NBVσNS through the Uniprot database.Thirty-one proteins were involved in cellular biological processes;thirty-six proteins were cellular anatomical components;thirty-one proteins had binding functions.Five proteins were part of the mitochondrial respiratory chain;seven proteins were ribosomal proteins and components of the ribosomal subunits;two proteins were involved in iron metabolism homeostasis.The GO functional enrichment analysis results showed that the interacting proteins were enriched in biological processes(BP)such as cellular processes,metabolic processes,biological regulation,l

关 键 词：纳尔逊海湾正呼肠孤病毒 酵母双杂交实验 σNS蛋白 蛋白互相作用 生物信息学 

分 类 号：R373[医药卫生—病原生物学]