Identification Of Duck Host Proteins Interacted With NS1 Protein Of Avian Influenza Virus Subtype H5N6

The Avian influenza virus(AIV)causes highly contagious acute infectious diseases in humans and animals and is classified as a notified disease by the World Organization for Animal Health(OIE).The nonstructural protein 1(NS1)regulates the infection process of influenza virus by interacting with various host proteins in the cell and is a key factor in regulating the pathogenicity of AIV.To identify host proteins that interact with the NS1 protein of AIV H5N6 subtype,this study: 1.Construction of Strep tag H5N6 recombinant AIV.Eight gene fragments of HN109 virus were amplified by RT-PCR and ligated into pBD bidirectional transcription expression vector.The amino acid 77-84 of the NS1 gene was replaced by the Strep tag by point mutation technique.The 8 recombinant plasmids were co-transfected into 293 T cells to rescue the recombinant virus r HN109-NS1-Strep.After sequencing verification,the 8 gene fragments that rescued were identical as the design.The Strep tag was verified being stably passed on until F5 generation based on both sequencing and protein expression level.Recombinant strains and wild strains showed similar replication in both MDCK cells by growth curve experiment,and in mice by viral pathogenic test.2.Isolation,culture and identification of duck primary lung epithelial cells.Lung primary cells were isolated by tissue dispersing from 24 d SPF duck embryos and purified by differential digestion.After three generations of purification,target epithelial cell markers,the cadherin and broad-spectrum keratin were detected by immunofluorescence,showing > 90% of cells positive and therefore identified as epithelial cells.3.Identification of NS1 interacted duck proteins.Recombinantion virus and wild virus respectively infect duck lung epithelial cells(MOI=5).After 24 h of infection,cells were lysed and centrifuged at 4 ?.The supernatant was purified by Strep resin for NS1 interaction proteins.The purified samples were analyzed by Western-blotting and mass spectrometry.A total of 36 host proteins interacting with NS1 were identified.