Establishment Of Extracellular Porcine ISG15 Sandwich ELISA Method And Preliminary Study On The Regulation Of PRRSV Replication

Porcine reproductive and respiratory syndrome(PRRS)is a swine infectious disease,which appeared in United States in 1987.Two years later,it was reported in in European regions and then rapidly spread to the rest world which results in huge economic losses.The pathogen causing PRRS is porcine reproductive and respiratory syndrome virus(PRRSV).Except attenuated vaccine,there is no effective method for the prevention,control and eradiation of PRRS.Therefore,further investigation is required to understand viral-host interaction of PRRSV with hosts.During pathogen invasion,the host will protect the host through innate immune response and adaptive immune response.As the key component of host innate immune response,of the host,type I interferons(IFNs)binds to its receptors on the cell surface and to activate the cells entering the antiviral state by inducing hundreds of interferonstimulating genes(ISGs).Among these ISGs,ubiquitin-like protein ISG15 is one of the ISGs with the fastest induction and the highest expression during IFNs stimulation.Similar to ubiquitin,ISG15 can covalently conjugated to the protein targets through cascade enzyme reaction.However,ISG15 can be released to extracellular space as monomer as a cytokine like protein.It has been reported that extracellular ISG15 could function as cytokines to stimulate the proliferation of NK cell,production of IFN-γ and maturation of DC,as well as chemokine for neutrophils chemo-axis.However,as an intracellular antiviral protein,the role of extracellular ISG15 on the immune system and its mechanism in viral infection is still unknown.In this study,the porcine ISG15 protein was recombinantly expressed and used for development of both monoclonal antibodies and polyclonal antibodies to establish sandwich ELISA of porcine ISG15.Further investigation was conducted to explore the interaction between PRRSV and ISG15,as well as the regulatory effect of ISG15 as a cytokine for PRRSV infection in PAMs.Our results were listed as follows:1.Development and characterization of monoclonal antibody against porcine ISG15.The prokaryotic expression vector p ET28a-ISG15 was constructed and the Hislabeled recombinant porcine ISG15 protein was purified from E.Coli.After immunization of Balb/c mice by porcine ISG15,the spleenocytes were collected for cell fusion,and a hybridoma cell line(Clone No.3D5E6)stably secrets antibodies recognizing porcine ISG15 antibody was obtained after ELISA and Western blot screening as well as and subcloning.The ascites for 3D5E6 was obtained by intraperitoneal injection of hybridoma cells to mice.After purification,the the antibody type of 3D5E6 was determine as Ig G1 using monoclonal antibody isotyping kit.Western blot and IFA showed that 3D5E6 could recognize endogenous expressed ISG15 in CRL2843 cells stimulated by porcine IFN-α.2.Establishment of sandwich ELISA for porcine ISG15.Immunization and purification of anti-porcine ISG15 polyconal antibodies was conducted by Genscript Ltd.Co(Nanjing,China).The mouse monoclonal antibody 3D5E6 and rabbit antiporcine ISG15 polyclonal antibodies was applied as capture antibody and detection antibody for ELISA,respectively.The recombinant porcine ISG15 protein was used as standard for sandwich ELISA procedure optimization,determination of detection limit of porcine ISG15 sandwich ELISA and the standard curve construction.The optimized detection conditions of sandwich ELISA are 8μg/well for capture antibody coating,the 1 to 4000 fold dilution of the detection antibody,and the lowest detection limit of porcine ISG15 is 250pg/m L.3.The difference of ISG15 expression level in PAMs infected by PRRSVs strains with different virulence.PAMs were infected with different PRRSV strains(with different virulence)for 24 hours,and the cells were harvested to evaluate the transcription level and protein level of porcine ISG15.Meanwhile,the supernatant of PAMs in different groups were collected as well and existence of porcine ISG15 was examined by porcine ISG15 sandwich ELISA.Moreover,VERO cells(IFN deficiency)were pretreated with the supernatant of PAMs infected with different PRRSV strains for 12 hours,followed by infection of IFN-sensitive NDV-GFP virus to detect the activity of IFNs in supernatant for 24 hours post inoculation.The result showed no difference in the number of GFP positive cells,which suggested that induction of ISG15 in PAMs infected by low virulence strain or vaccine strain of PRRSV was independent of IFN production.4.The regulatory effect of ISG15 protein on the replication of PRRSV on PAMs.The purified porcine ISG15 protein and the control protein SUMO were treated to remove endotoxin,and protein degradation was evaluated by SDS-PAGE.PAMs were pretreated with either porcine ISG15 or SUMO for 24 hours,and then infected with PRRSV-JXA1 strain for another 24 hours.Replication of PRRSV was determined by evaluating expression of N protein by Western blot.Compared with the control group,ISG15 protein could effectively inhibit PRRSV replication after stimulating PAMs.In conclusion,the attenuated strain and vaccine strain of PRRSV could induce higher level of ISG15 than virulence strains of PRRSV in PAMs.Moreover,ISG15 induced by PRRSV vaccine strain can be secreted or released extracellularly as cytokines,and the extracellular ISG15 protein can inhibit the proliferation of PRRSV in PAMs,implying that induction and secretion of porcine ISG15 in PAMs is related with PRRSV virulence in vivo.Our study provides new insight to understand the PRRSV virulence and pathogenesis for future prevention and control.