Effect Of Viperin Protein On Inhibiting Budding Of Duck Tembusu Virus In BHK-21 Cells And Establishment Of Cell Line Expressing Viperin

Viperin(virus inhibitory protein,endoplasmic reticulum-associated,IFN-inducible)is an interferon-inducible protein factor with antiviral effect.Duck Tembusu virus(DTMUV)belongs to the flavivirus of flaviviridae,which mainly causes the drop of egg production and the death of ducklings.To reveal the mechanism of Viperin protein action on DTMUV,we constructed a stable BHK-21 cell line expressing Viperin gene,and explored the effect of Viperin protein on DTMUV replication and budding at the cell level.1.Expression of Viperin and preparation of polyclonal antibody against Viperin proteinFirstly,Phylogenetic tree based on Viperin was constructed with Viperin gene of the different animals loaded down from Gen Bank.Then,the duck Viperin gene was cloned into p GEX-6P-1 vector and prokaryotic expression of Viperin fusion protein was carried out.Polyclonal antibody against duck Viperin was obtained by immunizing a rabbit with the purified Viperin fusion protein,1:8000 titer of polyclonal antibody was determined by indirect ELISA.2.Inhibitory effect of Viperin on budding of duck Tambusu virus in BHK-21 cellsRecombinant eukaryotic plasmid p EGFP-N1-Viperin was constructed and transfected into BHK-21 cells,and Viperin protein was expressed in BHK-21 cells.Then,p EGFP-N1-Viperin and p EGFP-N1 were transfected into BHK-21 cells respectively,followed by being infected with DTMUV.The copy number of virus was determined in the cell precipitate and supernatant by RT-q PCR 36 h post-infection.BHK-21 cells overexpressing Viperin protein were collected 36 h post-infection with DTMUV,followed by mass spectrometry analysis using EGFP monoclonal antibody capture.The results showed that DDX3 X and DDX5 related to DTMUV replication,four VAMP-3 proteins related to vesicular proteins,vesicular transporter SEC22,vesicular transporter GOT1 B and VAPA were captured.It was confirmed that Duck Tembusu virus was inhibited from sprouting in BHK-21 cells.3.Construction of BHK-21 cell line stably expressing duck derived Viperin proteinFirstly,p LOV-e GFP-Viperin vector expressing Viperin protein was constructed for preparing lentivirus-like particles,by co-transfecting 293 T cells with p SPAX2 and p MD2.G.The culture supernatant of 293 T cells containing lentivirus-like particleswas used to infect BHK-21 cells for consecutively passage.After passaging 20 generations,the stable cell line screened by puromycin pressure was determined by PCR,IFA and Western-blotting.Results showed that the constructed BHK-21 cell line stably express Viperin protein.To sum up,this study provided the technical support and academic reference for further exploring the mechanism of Viperin protein on restricting the budding of DTMUV.