人H5N1亚型禽流感病毒安徽株NS1基因的克隆及在原核系统的表达

Genetic analysis of NS1 fragment of human H5N1 Influenza virus isolated in Anhui province and its expression in Escherichia coli

摘要:

利用RT-PCR方法,从人H5N1亚型禽流感病毒安徽株扩增到了NS1基因,对其进行了克隆、序列测定和分析,并在原核系统高效表达和纯化了NS1蛋白。进化分析表明,A/Anhui/01/2005毒株与近些年国内分离的水禽H5N1病毒进化关系更为接近。NS1与福建、湖南分离的禽流感病毒同源性最高,分别达到99.1%和98.2%。序列分析表明,与病毒的致病性相关的92位氨基酸为Asp,与病毒的细胞因子抗性相关的80~84位氨基酸发生缺失,与断裂/多聚腺苷酸化特异性因子结合的基序改变为GFEWN,和病毒致死性相关的PL基序为ESEV。随后在大肠杆菌高效表达并纯化了NS1蛋白。NS1基因及其编码产物的特性分析以及在原核系统的表达,为进一步研究NS1的致病机制和抗病毒药物研制奠定了基础。

Abstract:

NS1 gene was amplified from an H5N1 influenza virus, A/Anhui/01/2005 for cloning, sequence analysis and later expression in Escherichia coli. The result showed that NS1 of A/Anhui/01/2005 strain had the close phylogenetic relationship with that of H5N1 avian influenza strains recently isolated in Fujian and Hunan. Correspondingly, its amino acid sequence showed the highest homology with that of Fujian and Hunan strains. The amino acid position of 92 involved in virus virulence was Asp, contrast to Glu in A/HK/156/97. Five amino acid deletion from 80 to 84 was also found in A/Anhui/01/2005, which was considered as a contributor to virus resistance against cytokine,such as IFN,TNF,etc. A motif binding to CBSF，conveted to GFWEN, which was different from previous GLEWN found in other 19 strains. Besides, the PL motif of ESEV, binding to PDZ domain,is the same as previous high-mortality H5N1 isolates. Furthermore, this NS1 was efficiently expressed in Escherichia coli and the highly purified product demonstrated wonderful activity as confirmed by Western blot. As a result，the work pays the way for further understanding the role of NS1 in human H5N1 infection and development of new antiviral drugs against influenza virus.