H7N9亚型禽流感病毒非结构蛋白1(NS1)基因序列分析及2013上海分离株NS1的原核表达

目的:对2013年3月发生的感染人的新型H7N9亚型禽流感病毒的非结构蛋白1(NS1)基因序列进行同源性分析,构建NS1重组质粒并表达。方法:从GenBank获得2006~2013年不同来源的H7N9亚型病毒NS1序列,并进行同源性比较;利用PCR方法从H7N9亚型禽流感病毒株A/Shanghai/4664T/2013(H7N9)基因组cDNA中扩增得到全长NS1基因,并将该片段定向克隆到原核表达载体pET28a上,构建重组质粒pET28a-NS1,经酶切鉴定,将重组质粒转化大肠杆菌BL21(DE3)感受态细胞后,IPTG诱导表达,且进行Western印迹分析。结果:经序列分析,2013年暴发的H7N9型禽流感病毒的NS1基因核苷酸序列同源性为95%~100%,与之前暴发的H7N9型流感病毒NS1基因序列的同源性为86.4%~90.7%,表明2次暴发的该型流感分离株属于不同的进化分支;PCR扩增得到约680 bp的NS1基因序列,所克隆的NS1基因在原核细胞中的表达产物主要以包涵体形式存在,SDS-PAGE检测结果表明重组蛋白相对分子质量为25×103,Western印迹分析证实表达产物为H7N9禽流感病毒NS1蛋白。结论:为进一步研究H7N9亚型流感病毒NS1蛋白功能及基于NS1蛋白的抗病毒药物奠定了基础。

Gene Sequence Analysis of NS1 Protein from Avian Influenza Virus H7N9 Subtype and Prokaryotic Expression of NS1 Protein from Shanghai Strain

Objective： To analyze the sequence homology of the NS1 gene in a novel avian-origin influenza H7N9 virus（first identified in March 2013）, and express and purify NS1 protein. Methods： We analyzed the nucleotide sequence homology of the NS1 gene of H7N9 subtype avian influenza which caused outbreaks from 2006 to 2013. The full length of NS1 gene of the human influenza A virus A/Shanghai/4664T/2013（H7N9） strain cDNA was amplified by PCR, then cloned into prokaryotie expression vector pET28a. The pET28a-NS1 was identified by restriction endonuclease digestion and transformed into E.coli BL21（DE3）. Then NS1 protein expression was induced by IPTG and detected with SDS-PAGE and Western blotting. Results： Based on bioinformatic analysis showing 95% to 100% nueleotide sequence homology of the NS1 gene of H7N9 avian influenza virus exists among dif- ferent strains of novel H7N9 virus from 2013, while 86.4%-90.7% homology of earlier strains of avian influenza virus, we presumed that H7N9 subtype influenza outbreak in 2013 and in previous years belongs to different evolution branches. The cloned 680 bp gene was identified as NS1 gene by sequencing. Then, we obtained NS1 pro- tein, and most of which existed in the form of an inclusion body. SDS-PAGE and Western blotting indicated that the relative molecular weight of expressed product was about 25-103 and the expressed protein was NS1 protein of H7N9 subtype avian influenza. Conclusion： Providing a foundation to help further studies on the function of NS1 gene and in preparation of antibodies to NS1 protein.