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.
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.
龙彩韵;聂凯;王环宇;王瑞晨;姚晓慧;武婕慧;付士红;李樊;殷启凯;崔倩倩;许松涛
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中国疾病预防控制中心病毒病预防控制所,北京 102206
基础医学
乙型脑炎病毒;MS2噬菌体;假病毒
Japanese encephalitis virus;bacteriophage MS2;pseudovirus
《中国人兽共患病学报》 2024 (003)
231-235,242 / 6
国家重点研发计划(No.2020YFE0205700,No.2022YFC2302700) Supported by the National Key Research and Development Program(No.2020YFE0205700,No.2022YFC2302700)
10.3969/j.issn.1002-2694.2024.00.043
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