H9N2亚型禽流感病毒ns1基因的融合表达

Fusion Expression of ns1 Gene of Avian Influenza Virus H9N2 Subtype in E.coli BL 21(DE3)

禽流感病毒(AIV)非结构蛋白NS1在病毒粒子中不存在,因此,可以通过检测NS1抗体来鉴别禽流感病毒感染鸡群和免疫鸡群.将ns1基因和T4噬菌体soc基因在大肠埃希氏菌中融合表达,融合蛋白可以作为鉴别禽流感免疫鸡群和感染鸡群的检测抗原.采用RT-PCR方法扩增禽流感病毒H9N2的ns1基因(654 bp);将该片段克隆至pSOC质粒soc基因3′端,成功构建了重组质粒pSOC-NS1,用该重组质粒转化E.coliB l21(DE3)感受态细胞,以终浓度1 mmol/mL的IPTG诱导表达.SDS-PAGE结果表明pSOC-NS1融合蛋白相对分子质量约39 000.W estern-b lot证实,表达产物与AIV H9N2病毒感染的小鼠血清具有良好的反应性.

Non-structual protein （ NS1 ） of avian influenza virus （AIV） does not exist in virion,so it could be used to detect NS1 antibody for the differentiation of infected flocks from vaccinated ones. In the present study,ns1 gene was expressed fusing with soc gene of T4 bacteriophage in E. coli, and fusion protein could be used as antigen to detect antibody against NS1 protein. The ns1 gene with a length of 654 bp was amplified by RT-PCR from AIV subtype H9N2. The ns1 fragment was inserted into plasmid pSOC to obtain recombinant plasmid pSOC-NS1 in which ns1 gene was fused into the 3＇-teminal of soc gene. pSOC-NS1 was transformed into E. coli BL 21 （DE3） to induce ns1 gene expression with 1 mmol/L IPTG. A NS1 fusion band with relative molecular mass of appoximately 39 000 was observed on the SDSPAGE gel and nitrocellulose membrane. Western-blot analysis confirmed that the fusion protein could specifically react with the mouse sera infected with live avian influenza virus H9N2 subtype.