Identification Of Cell Proteins Interacting With PRRSV Nsp4

Porcine reproductive and respiratory syndrome(PRRS)is one of primary infectious disease that threatened global pig industry,characterized by skin cyanosis,reproductive disorder in sows,espiratory problems in pigs of all ages,and high lethal rate of piglet.The virus also in fected pregnant sows causing persistent infection.This disease is also called "Blue Ear".Porcine reproductive and respiratory syndrome virus(PRRSV)is its pathogen.The PRRSV genome which belongs to the positive-strand RNA viruses codes for seven structural proteins and 14 nonstructural proteins.Nsp4 contains serene protease,accounting for cleaving nsp3-nsp12.In order to study the nsp4 role in the process of PRRSV infection,This study obtained the puri fied nsp4 protein expressed by Escherichia coli.The purified recombined nsp4 were used to i mmunize New Zealand rabbits,and polyclonal antiserum has good specificity reaction.In or der to analysis the proteomics of nsp4,we process the Western blot,,Laser confocal method,co-immunoprecipitation to verified interrelationship between nsp4 and Myosin IIb(MYH10),SRCAP and TRIM28 after Mass spectrometry.The details as follow: 1.Preparation and iden tification of antibody to nsp4 of PRRSV: to detection of nsp4 antibody in porcine reproductive and respiratory syndrome virus(PRRSV)infected pigs.Nsp4 gene of highly pathogenic PRRSV was amplified and cloned into pET-28a(+)vector,designated pET28a-nsp4.The soluble fusion protein expressed from pET28a-nsp4 was purified.The anti-serum against nsp4 w as obtained by immunizing new zealand rabbits using the purified nsp4.The value of polyclonal antibody was about 106 detected by ELISA.Immunofluorescence assay and Western-blot analysis showed that polyclonal antibody has a good specificity and can recognize the native protein in Marc-145 cells infected by PRRSV;2.Study on proteomics of nsp4: in this study,PRRSV nsp4 genes were cloned into pEGFP-C1,The recombinant plasmid pEGFP-C1-nsp4 was transfected into 293 T cells to express recombinant GFP-nsp4 protein.By pulldown assay,GFP-Trap beads was used to precipitate the cellular proteins interacting with nsp4.The pull down product was sented to mass spectrometry after verifying the successful operation of the pulldown experiment by western blot,We obtained a total of 45 cellular proteins interacting with nsp4;3.Screening and identification of nsp4 interacting protein: three kinds of cell protein MYH10,SRCAP,TRIM28,which are highly interactive with nsp4,were screened out in the results of mass spectrometry analysis.The results of western blot showed that MYH10 and TRIM28 existed in pulldown products;MYH10,SRCAP and TRIM28 antibody were used to co immunoprecipitation,respectively.The results showed that GFP-nsp4 fusion protein was present in CO-IP;Laser confocal showed that MYH10,SRCAP,TRIM28 and nsp4 had different degrees of co-localization,and MYH10,SRCAP,TRIM28 and PRRSV also had significant co-localization.In conclusion,PRRSV nsp4 interacts with MYH10,SRCAP and TRI M28 three cell proteins.