H9N2亚型AIV NS1基因的克隆及其在杆状病毒系统的表达

Molecular Cloning of Non-Structural Protein NS1 Gene of H9N2 Avian Influenza Virus (AIV) and Its Expression in Insect Cells

应用DNAStar软件,参照Genbank中注册的AIV H9亚型毒株NS1基因序列,设计了一对引物,用RT-PCR方法成功地扩增出带双酶切位点的H9亚型AIV的NS1基因,通过BamHⅠ和EcoRⅠ双酶切位点将NS1基因插入转移质粒载体pFastbacHTa中,获得重组转移载体pFastbacHTa-H9NS1并将其转化DH10Bac细胞,与Bacmid发生位点特异性转座作用,得到重组穿梭载体Bacmid-H9NS1,再将其转染昆虫细胞sf9,PCR鉴定证实该基因正确地插入到病毒基因组的多角体蛋白基因启动子下游,经过SDS-PAGE和WesternBlot检测,H9 NS1基因在sf9细胞中得到了表达,H9 NS1大小约为28kD,而且表达的产物具有特异免疫学反应性。

A pair of primers was designed by employing DNAStar ,software and consulting the NS1 nucleotide sequences of HgN2 subtypes AIV. Then the NS1 gene with flanking restriction sites was successfully PCR-amplified. The Hg-NS1 gene was excised with BamH Ⅰ and EcoR Ⅰ and inserted into the transfer vehicle pFastbacHTa to obtain the recombinant transfer vectors pFastbacHTa-H9NS1, which was used to transform DH10 Bac-host cells, producing specific transposition with Bacmid to yield the recombinant shuttle vehicles, Bacmid-HgNS1. And then the Bacmid-HgNS1 was further used to transfect sf9 insect cells, when PCR identification proved that the gene was inserted in correct orientation downstream of the virus polyhedral protein promoter; SDS-PAGE and Western blotting detection confirmed that the gene had been expressed in sf9 cells, the size of the H9NS1 being approximately 28 KD. The expression product could specially react to the sera of AIV.