人星状病毒I型非结构蛋白nsP1a及其C末端蛋白nsP1a/4的原核表达及鉴定  

Prokaryotic Expression and Identification of Human Astrovirus Nonstructural Proteins,nsP1a and nsP1a/4

作　　者：刘文慧[1] 阚丽丽 崔永胜[3] 谭李倩 梁雪雪 李新[1] 赵微[1] 

机构地区：[1]辽宁医学院,锦州121200 [2]盘锦市辽河油田总医院,盘锦124000 [3]山东省东营市胜利油田中心医院,东营257034

出　　处：《病毒学报》2015年第1期46-50,共5页Chinese Journal of Virology

基　　金：国家自然科学青年基金项目(No.81201285);辽宁省博士启动基金项目(No.20141134);辽宁医学院校长基金-奥鸿博泽基金大学生创新项目编号(NO.2014D09)

摘　　要：人星状病毒(Human Astrovirus,HastV)是导致婴幼儿腹泻的重要病原体。HastV非结构蛋白nsP1a及C末端蛋白nsP1a/4含有各种保守的功能结构域,在星状病毒的复制、转录,病毒与宿主的相互作用中起重要作用。为获得nsP1a及其nsP1a/4蛋白,为后续蛋白相关研究提供平台,本研究在E.coli系统中进行人星状病毒非结构蛋白nsP1a及nsP1a/4蛋白的表达并对表达产物进行鉴定。首先将nsP1a及nsP1a/4基因克隆入原核表达载体PGEX-4T-1,构建nsP1a及nsP1a/4蛋白融合表达质粒;在E.coli BL21(DE3)中进行IPTG诱导表达,摸索两种融合蛋白表达的最优条件并对表达蛋白进行免疫印迹鉴定。结果表明nsP1a蛋白在30℃,1mM IPTG诱导12h时,蛋白表达量达到最高;nsP1a/4蛋白在20℃,0.5mM IPTG诱导8h时,蛋白表达量达到最高。Western blot结果显示两种融合蛋白既可与nsP1a蛋白免疫血清发生特异性反应,也可被GST标签抗体所识别。本研究成功利用原核系统表达并鉴定了人星状病毒非结构蛋白nsP1a及其C末端蛋白nsP1a/4,为进一步研究星状病毒非结构蛋白的功能及病毒的致病机制奠定基础。Human astrovirus （HastV） is recognized as one of the leading causes of acute viral diarrhea in infants. The HastV non-structural protein, nsP1a, and C-terminal protein, nsP1a/4, contain various conserved functional domains, and may play an important role in virus replication, transcription and the virus-host interactions of HastV. This study used an E.coli system to investigate the expression of nsP1a and nsP1a/4 proteins. Firstly, the nsP1a and nsP1a/4 genes of HAstV-1 were cloned into the prokaryotic expression vector, PGEX-4T-1, to build the PGEX-4T-1a and PGEX-4T-1a/4 fusion protein plasmids. Then, the recombinant plasmids were transformed into Escherichia coli BL21 （DE3） and induced with isopropyl-β-D-thiogalactopyranoside （IPTG）. The optimal expression conditions of the two fusion proteins were identified and then analyzed by polyacrylamide gel electrophoresis （SDS-PAGE） and western blotting, respectively. The results showed that the pGEX-4T-1a fusion protein was maximally expressed at 30℃ after 12 hours of induction with 1.0 mM IPTG. The pGEX-4T-1a/4 fusion protein was maximally expressed at 20℃ after 8 hours of induction with 0.5 mM IPTG. Western blot analysis showed that the two fusion proteins specificity reacted with the anti-nsP1a and anti-GST monoclonal antibodies, respectively. This study successfully obtained the HAstV non-structural protein, nsP1a, and its C-terminal protein nsP1a/4 protein using an E.coli system. This novel study lays the foundation for future research into the pathogenic mechanisms of human astrovirus and the functions of its non-structural protein.

关 键 词：人星状病毒 非结构蛋白nsP1a nsP1a/4 原核表达 融合蛋白 

分 类 号：Q78[生物学—分子生物学]