Expression of the NS1 gene of H5N1 avian influenza virus in E.coli and insect cells

To construct the E.coli expression vector pET-NS1 and the baculovirus transfer vector pFast-NS1 for the expression of the NS1 gene of H5N1 avian influenza virus in E.coli and insect cells,the NS1 gene was inserted to the E.coli expression vector pET-28a(+)or baculovirus transfer vector pFast-NS1 after digestion of the plasmid pUC-NS1 to construct the recombinant plasmids pET-NS1 and pFast-NS1 respectively.E.coli BL21 strain was transformed with pET-NS1 by induction through IPTG,whereas the buculovirus-competent cell DH10 was transfected with pFast-NS1,and then the recombinant bacmid Bac-NS1 DNA was extracted and identified by PCR with M13 universal primers.The Sf9 cells were transfected with screened Bac-NS1 by lipofectin,and the transfected cells were collected 72 hours after transfection.Meanwhile,the expressed product was analyzed with SDS-PAGE and Western blotting.In these ways,the E.coli expression vector pET-NS1 and the baculovirus transfer vector pFast-NS1 were successfully constructed,and specific bands of fusion proteins could be demonstrated in Western blot analysis.It is apparent that the NS1 gene can be expressed correctly in E.coli and insect cells with production of specific NS1 protein,thus laying a foundation to obtain large amount of NS1 protein for the preparation of specific antibodies.