禽流感病毒B类等位基因NS1蛋白的原核表达、纯化及热稳定性分析  

Prokaryotic Expression, Purification, and Thermal Stability Analysis of the Allele B NS1 Protein of Avian influenza virus

作　　者：罗维玉[1,2] 胡永浩[1] 张杰[2] 王金光[2] 朱鹏阳[2] 梁立滨[2] 周圆[2] 李呈军[1] 姜丽[2] 陈化兰[1,2] 

机构地区：[1]甘肃农业大学动物医学院,兰州730070 [2]中国农业科学院哈尔滨兽医研究所/兽医生物技术国家重点实验室,哈尔滨150001

出　　处：《农业生物技术学报》2016年第10期1482-1490,共9页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金(No.31402206)

摘　　要：A型流感病毒的非结构蛋白基因(non-structural protein,NS)在进化上可以分为等位基因A和B,两者之间同源性低于70%;NS1是流感病毒对抗宿主免疫系统的主要毒力因子之一。本研究在序列比对分析的基础上,表达、纯化了本实验室保存的4株B类等位基因禽流感病毒(Avian influenza virus,AIV)(A/duck/Hunan/S1256/12(H3N8),A/environment/Hunan/S4484/11(H12N7),A/duck/Guangdong/07/00(H5N1)和A/duck/Shanghai/08/01(H5N1))的NS1蛋白,并对其进行稳定性分析。首先从感染的鸡胚尿囊液中提取病毒总RNA,利用特异引物Uni12反转录获得c DNA,以其为模板PCR扩增得到NS1全长片段,利用Bam HⅠ和NotⅠ双酶切将其定向插入p GEX6P-1载体,大肠杆菌(Escherichia coli)BL21菌株中诱导表达。该蛋白在纯化过程中稳定性差,沉淀析出。为此,进一步构建NS1蛋白截短突变体(1M-202A,R38A/K41A),并在BL21菌株中诱导表达,经过GE health Glutathione sepharose 4B亲和层析、Source Q阳离子交换柱层析和Hitrap Superdex75分子筛凝胶层析逐步纯化,然后进行SDS-PAGE凝胶纯度分析,并测定OD320,进行蛋白热稳定性分析。结果表明,经进化分析,4株病毒均属于禽流感病毒B类等位基因群,4株病毒NS1蛋白经过全长野生型、全长突变体及截短突变体的逐步优化表达,截短突变体NS1蛋白表达质量相对较高,最终经过逐步纯化获得纯度达到90%以上的蛋白样品,且4株病毒中A/duck/Hunan/S1256/12(H3N8)NS1蛋白的稳定性最好。研究结果为进一步研究其分子结构与功能提供了基础资料。The non-structural protein gene（NS） of influenza A viruses is separated into 2 alleles, A and B,with less than 70% sequence homology between each other. The NS1 is one of the major virulent factors that are able to antagonize the host immune system. In this study, on the basis of a systemic phylogenetic analysis,the NS1 protein of 4 Avian influenza viruses（AIV） strains（A/duck/Hunan/S1256/12（H3N8）, A/environment/Hunan/S4484/11（H12N7）, A/duck/Guangdong/07/00（H5N1） and A/duck/Shanghai/08/01（H5N1）） with NS gene of allele B prokaryotically expressed and were purified, and their thermal stability were furtherexamined. Viral RNA（v RNA） was extracted from virus-infected allantoic fluid, followed by c DNA synthesis by reverse transcription PCR with the Uni12 primer. The full- length of NS1 fragments were then amplified with specific primers and cloned into the p GEX6P-1 vector for expression in the BL21 bacterial strain. Since the wild- type NS1 proteins were unstable and precipitated during purification, we determined to generate truncation mutants of NS1 protein containing amino acids 1M- 202 A as well as two mutations, R38A/K41 A.The truncation mutant constructs expressed in BL21 bacterial strain, and progressively purified by Glutathione sepharose 4B affinity chromatography, cation exchange chromatography, and Hitrap Superdex75 column chromatography. The phylogenetic analysis revealed that the NS1 protein of all 4 viruses belonged to allele B. The expression and purification condition of NS1 protein was sequentially optimized from wild type,full length mutant to truncated mutant. High quality of purified NS1 protein with purity over 90% was finally obtained from the truncated mutant. In addition, the NS1 protein of A/duck/Hunan/S1256/12（H3N8） exhibited the highest heat stability. This study provides a foundation for further studying the structure and biological function of the allele B NS1 protein.

关 键 词：禽流感病毒 NS1蛋白 原核表达纯化 热稳定性 

分 类 号：S855.3[农业科学—临床兽医学]