Screening The Interactome Of Porcine Epidemic Diarrhea Virus Nucleocapsid Protein

Porcine Epidemic Diarrhea(PED)is an acute contact intestinal infectious disease characterized by diarrhea,vomiting and dehydration in suckling piglets.The mortality rate of the nursing piglets within one week is almost 100%,and bring the huge economic losses to the global pig industry.That caused by infection with the porcine epidemic diarrhoeal virus(PEDV).For the encapsulated single-stranded positive-strand RNA virus,it encodes seven open reading frames.The N protein encoded by the N gene plays an important role in the process of viral transcriptional replication,and can enter the nucleus by localization signals and participate in the regulation of nucleolus.In order to better understand the biological function of N protein,explore the role of N protein in PEDV replication,and the pathogenesis of PEDV,We study the interaction proteomics of N protein and obtained interaction with N protein.The cellular protein provides a theoretical basis for further screening for drug targets that inhibit PEDV infection.In order to obtain the cellular protein of N protein interaction,the PEDV N target fragment was amplified by using the cDNA of PEDV PEDV LJX01/2014 as a template,and the target fragment was ligated with pEGFP-C1 eukaryotic expression vector to construct pEGFP-N recombinant plasmid.After transfecting the recombinant plasmid into 293 T cells,the pEGFP-N protein and its interacting cell proteins were specifically precipitated by the Pull-down assay.The Pull-down assay was successfully used to confirm the pEGFP-N protein and the remaining samples were sent by Western blot.Mass Spectrometry a total of 125 cellular proteins interacting with the pEGFP-N protein were screened.Four kinds of cellular proteins DHX9,NCL,KAP1 and TCEA1,which have high possibility of interaction with pEGFP-N protein,were screened out from the results of mass spectrometry,and their interactions were confirmed by Western blot,co-immunoprecipitation and laser confocal experiments.Western blot analysis showed that DHX9,NCL,KAP1 and TCEA1 were present in the Pull-down products of 293 T and Vero E6 cells.DHX9,NCL,KAP1 and TCEA1 were used in 293 T and Vero E6 cell lysates overexpressing pEGFP-N protein,were co-immunoprecipitation experiments,and pEGFP-N fusion protein was detected in each product;PEDV N monoclonal antibody and HX9,NCL,KAP1 and TCEA1 were used in Vero E6 cells infected with PEDV,respectively.Antibodies were positively and negativelyco-immunoprecipitated and their respective interacting proteins were detected;laser confocal microscopy indicated colocalization of NCL and pEGFP-N in 293 T and Vero E6 cells.Therefore,it was concluded that the interaction proteomics of the PEDV N protein was successfully obtained,and the cellular protein NCL interacted with the N protein.This study laid the foundation for further study of the pathogenesis of PEDV.