Screening And Verification Of Interacting Proteins Of H5N1 Avian Influenza Virus NS1

NS1?Non-Structural protein?,the avian influenza virus,is a highly expressed protein when the influenza virus infects the host cells.But NS1 does not exist in mature viruses.Therefore it is called a non-structural protein.NS1 plays an important role in the process of influenza virus infection of host cells.The main roles are to resist the immune response of hosts and prevent the production of interferon to further antagonize the viral defense of interferon formation.In addition,NS1 is also an important virulence factor in avian influenza viruses.Its key sites can affect the pathogenicity of influenza viruses.NS1 also regulates the cleavage and nuclear-mass output of mRNA,which is very helpful for viral infection and replication.NS1protein can interact with various proteins to alter the intracellular distribution of host proteins and regulate the virulence and pathogenicity of viruses.A recombinant plasmid with GST tag was constructed using the NS1 gene of H5N1 avian influenza virus to further study the role of NS1 protein in replication and pathogenesis of avian influenza virus.The conditions of protein expression have been optimized.Then high-purity fusion protein was used for GST-Pull down.More proteins interacting with NS1 was screened in human lung adenocarcinoma cell line A549 with using GST protein as a control.Therefore to verify the interaction between potential protein and NS1,two proteins were prokaryotically expressed.ELISA technology was used to verify the interaction between the two proteins.After eukaryotically expressing two proteins,Co-IP technology was used in order to verify the mutual interaction of the two proteins.The results showed that in the GST-NS1 experimental group there were two different bands compared to the GST control group.By mass spectrometry?MAODI-TOF?,a positive result was got from one of the bands.The interaction protein is?-actinin 4,and its interaction with NS1 is still not reported.The two proteins were prokaryotically expressed.The interaction between NS1 and?-actinin 4was confirmed by ELISA.The results showed that the absorbance value of OD_450nm50nm in the experimental group was positively correlated with the concentration of NS1compared to the control group.The results and analysis indicating that the NS1protein can bind to the coating antigen?-actinin 4.The co-immunoprecipitation experiment was carried out by co-transfecting the Flag-His-ACTN4 plasmid with the HA-NS1 plasmid into 293T cells.The results further confirmed the interaction between NS1 and?-actinin 4.This study laid the foundation for exploring the pathogenic mechanism of NS1 in avian influenza virus.