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 .