鸭源H9N2亚型流感病毒NS基因的克隆及表达

根据GenBank中收录的H9N2亚型流感病毒的NS基因序列设计合成了 1对引物NSU/NSL ,利用RT PCR扩增出了H9N2株NS基因 ;将该基因片段克隆到 pMD 18 T载体上 ,并对所得到的重组质粒进行酶切分析及序列测定。结果 ,获得了NS基因片段的阳性重组子 ,扩增的NS基因包含NS1基因完整的阅读框架和部分NS2基因 ,其序列与GenBank中收录的其他分离株NS基因比较 ,同源性达 96 %～ 99%。再将克隆的NS基因插入到原核表达载体 pET 2 8a后 ,转化E .coliBL2 1(DE3)感受态细胞 ,在IPTG诱导下获得了预期的蛋白表达 ,所表达蛋白质的分子质量约为 30ku。

Cloning and expression of NS gene of avian influenza virus type H9N2 from ducks

With one pair of primers which were designed and synthesized according to the sequence of nonstructural protein (NS) gene published in GenBank, a fragment 820 bp in length was amplified from (avian) influenza virus type H9N2 from ducks by RT-PCR. Then the fragment was cloned into the pMD 18-T vector to construct a recombinant plasmid. The constructed recombinant plasmid was proved to be true by restriction endonuclease analysis. In order to further identify the credibility of the gene, the sequence of the gene's cDNA was obtained by sequencing technique. It was confirmed that the nucleotide sequence (amplified) in the experiment shared 96%-99% homology with the corresponding sequences published in (GenBank), and comtained the NS1 gene's ORF and the NS2 gene's part sequence by means of sequencing. (Afterwards), the NS gene fragment was cloned into the expression vector pET-28a to construct the recombinant plasmid pET/NS. The constructed recombinant plasmid pET/NS was transformed intoE.coli BL21 (DE3) competent cells and induced with IPTG. Finally, the target protein was expressed and its molecular weight was 30 ku as expected by us .