乙型脑炎病毒部分NS1基因假病毒的构建
Construction of pseudoviruses containing a partial NS1 gene of Japanese encephalitis virus
龙彩韵 1王瑞晨 2姚晓慧 2武婕慧 2付士红 2李樊 2殷启凯 2崔倩倩 2许松涛 2聂凯 2王环宇2
作者信息
- 1. 安徽理工大学医学院,淮南 232001;中国疾病预防控制中心病毒病预防控制所,北京 102206
- 2. 中国疾病预防控制中心病毒病预防控制所,北京 102206
- 折叠
摘要
目的 构建耐核酸酶的含乙型脑炎病毒(Japanese encephalitis virus,JEV)部分非结构蛋白1(Non-structural protein 1,NS1)基因的假病毒,为JEV的实验室核酸检测提供质控品,提高检测质量.方法 将JEV部分NS1基因、蚊18S RNA基因和人GAPDH基因片段次序连接克隆到该质粒上,构建重组载体pET-MS2-JEV;转化大肠杆菌后表达纯化假病毒,并对其进行耐受性和稳定性评估.结果 构建了重组载体pET-MS2-JEV,表达获得的假病毒具有耐核酸酶、耐物理化学因子的特性,可在4 ℃和-20 ℃条件下稳定保存.结论 本研究获得了稳定性好的含JEV部分NS1基因的假病毒,可作为JEV实验室核酸检测的质控品.
Abstract
Construction of pseudoviruses containing a partial non-structural protein 1(NS1)gene of Japanese encephalitis virus(JEV)with nuclease resistance for application as quality control reagents in laboratory assay.Firstly,the plasmid pET-32a-MS2 underwent optimization and modification.Secondary,fragments of the partial NS1 gene of the JEV,along with inter-nal reference genes from mosquitoes and humans(mosquito 18S RNA and human GAPDH),were inserted into this plasmid,resulting in the construction of the recombinant plasmid pET-MS2-JEV.Subsequent steps involved the expression and purifica-tion of pseudoviruses.The construction of the recombinant plasmid pET-MS2-JEV yielded pseudoviruses,which obtained resil-ience against nucleases and various physical and chemical factors.These pseudoviruses exhibited stability when stored at various temperature conditions,including 4 ℃ and-20 ℃.This study accomplished the production of pseudoviruses characterized by outstanding stability,thus serving as reliable quality control reagents for JEV laboratory assays.
关键词
乙型脑炎病毒/MS2噬菌体/假病毒Key words
Japanese encephalitis virus/bacteriophage MS2/pseudovirus引用本文复制引用
基金项目
国家重点研发计划(2020YFE0205700)
国家重点研发计划(2022YFC2302700)
出版年
2024
国家科技期刊平台
NSTL
维普
万方数据