猪源流感病毒NS1基因的克隆及原核表达

对H3N2亚型猪流感病毒NS1基因进行原核表达，获得纯化表达产物，以期为检测猪流感抗体的ELISA试剂盒的研制奠定基础。采用RT-PCR方法扩增H3N2亚型猪源流感病毒的NS1基因（693bp），并将该片段克隆至pET-28（a）构建重组表达质粒pET-NS1。将重组质粒转化E．coli BL21（DE3）感受态细胞，以终浓度1mmol／L的IPTG在不同时间进行诱导表达，SDS-PAGE检测蛋白表达情况，并经Western-blot分析表达产物的抗原性。pET—NS1重组表达质粒表达的NS1蛋白相对分子质量约为26kD，1mmol／L的IPTG诱导4h时蛋白表达量达到高峰。Western-blot印迹分析证实表达蛋白能与阳性血清发生特异性反应，而与阴性血清不反应。NS1基因的原核表达产物可用来鉴别流感感染猪群和灭活疫苗免疫猪群。

Cloning and Prokaryotic Expression of NS1 Gene of H3N2 Swine Influenza Virus

The NS1 gene of H3N2 subtype influenza A virus was expressed in prokaryotic expression system to obtain the recombinant protein for the manufacture of ELISA detection kit for swine influenza virus. The NS1 gene of one H3N2 subtype swine influenza A virus was amplified by RT-PCR, and then cloned into prokaryotic expression vector pET-28a （＋）. The recombinant plasmid pET-NS1 was extracted after being transformed into E. coli BL21 （DE3） competent cells and was induced expression for NS 1 gene with 1 mmot/L IPTG at different times. The protein expression was assayed with SDS-PAGE and the protein immunity was analyzed by Western-blot. The NS 1 protein with relative molecular weight of approximately 26 kD could be highly expressed. The western-blot test showed that the recombinant protein could react with the positive sera, but not with the negative sera. The protein expressed in E.coli BL21 （DE3） could be applied to discriminate the influenza infected pigs from the vaccinated ones.