H5亚型禽流感病毒NS1基因的克隆及在原核细胞中的表达

Cloning and Expressing of Avian Influenza Virus NS1 Gene in Procaryotic Cell

以RT-PCR方法从H5N1亚型禽流感病毒A/DKZJ/12/00(H5N1)中获得禽流感病毒NS1的部分基因,将目的基因定向克隆到原核表达载体pET-32a,将测序和酶切验证正确的阳性重组质粒转化大肠杆菌BL21,经IPTG诱导表达,蛋白质电泳表明该蛋白得到了可溶性的表达,表达蛋白的分子量27 kD;Western-blot分析表明,该蛋白可以与禽流感H5阳性血清反应,具有良好的免疫原性。

The complementary DNA of NS1 gene was prepared from the cDNA by RT-PCR. The amplified fragment was cloned into the plasmid pMD18-T. The sequence coding of signal peptide M was deleted. The PCR products and the expressing vector were digested with restriction endonuclease EcoR I and Hind Ⅲ , then the NS1 gene was inserted into the pET-32a system, after that, the constructed recombinant plasmid was transformed into BL21（DE3）. The recombinant protein was expressed and identified by SDS-PAGE and Western blot,which proved it has good biological activity.