The Preliminary Research On Fuction And Mechanism Of Porcine ISG15 Against PRV

ISG15,the product of interferon stimulated gene 15(ISG15),plays a central role in the host’s antiviral response against many viruses.Of the hundreds of interferon stimulated genes(ISGs)discovered to date,ISG15 was one of the first identified and shown to encode a ubiquitin-like protein.Similar to ubiquitin,through an conjugation cascade,ISG15 is covalently linked to a variety of target proteins,in part,as a modifier of protein function.Mass spectrometric analysis has led to the identification of several hundreds of candidate proteins that can be conjugated by ISG15.Studies performed over the past several years have shown that ISG15 is associated with the antiviral activity and regulating interferon signaling.Only the antiviral activity of human and mice ISG15 s have been performed,that,at present,the function of porcine ISG15 is still unknown.We focus on cloning and expression of porcine ISG15 gene,obtain polyclonal antibodies,establishing a PK-15 cell line stably expressing porcine ISG15 and preliminary research on porcine ISG15 inhibits replication of pseudorabies virus(PRV)to explore the function of porcine ISG15 and expect which will be useful for developing a novel treatment to combat PRV infection and therapies targeting this pathway can be developed in the future.1.Expression of porcine ISG15 protein and polyclonal antibodies preparationThe ISG15 gene of porcine was amplified by RT-PCR from peripheral blood lymphocytes cells.The porcine ISG15 gene was cloned into p ET28 a and expressed with 6×histidine tag by IPTG and purified by Ni2+ affinity chromatography.Rabbits were immunized with purified ISG15 protein to obtain the polyclonal antibodies against this protein.The porcine ISG15 gene was found to consist of 504 bp encoding 167 amino acid residues.Compared with the known ISG15 of other species,the most conserved regions of the porcine ISG15 peptide were found to be the tandem ubiquitin-like domains(No.3-75 amino acid in the N-terminal and No.81-156 amino acid in the C-terminal)and LRLRGG conjugating motif in the C-terminal,characteristic of the ISG15 proteins.Western-blot analysis showed that the antibodies had good specificity.The ELISA results showed that the antibody titer of polyclonal antibodies was 1:102400.This study result lays a foundation for further revealing the function of porcine ISG15.2.Establishment of a PK-15 cell line stably expressing porcine ISG15The recombinant expression vector(Piggy Bac-p ISG15)was successfully constructed,and transfected into PK-15 cells using LipofectamineTM2000.The transfected PK-15 cells were screened by 5ug/m L puromycin selection for two weeks.The monoclonal cell was acquired by limiting dilution from the polyclonal mass of stably transfected cells to observe stable green fluorescence.A single clone of transfected PK-15 cells expressing porcine ISG15 was generated by several rounds of cloning and sub-cloning and identified by q RT-PCR assay and western blot detection,respectively.The results showed that porcine ISG15 expressed in stably transfected cell line after continuous passage culture,and the expression of ISG15 was increased extremely significantly(9-fold)compared with that in normal PK-15.The PK-15 cell line stably expressing porcine ISG15,named PI-15,provides a useful tool for the study on the other functions else of porcine ISG15 as well as on its mechanism.3.Inhibition of PRV replication by porcine ISG15To investigate the transcript pattern of PRV regulated by porcine ISG15,q RT-PCR and Plaque-forming unit assay were performed to examine the expression profiles of PRV EP0 m RNA and its titer in PI-15 cell at 12h、24h、36h and 48 h post-infection.The results showed that porcine ISG15 inhibits significantly replication of PRV during all infection,compared with mock-infected cells.The results of Plaque-forming unit assay showed that over-expression of porcine ISG15 in PK-15 cells significantly reduced the PRV plaque titer by 5-fold at 24 h post-infection.In addition,q RT-PCR assay indicated that the PRV EP0 m RNA level in the supernatant of infected ISG15-over-expressing cells was significantly lower than(5-fold)that in the infected vector control cells.When ISG15 gene of PK-15 cells was knocked down by si RNA which targeted the DNA conservative domain,no differences in viral growth of PRV have been displayed in ISG15-over-expressing cells and vector control cells during all infection.The results demonstrated that over-expression of ISG15 significantly inhibited PRV replication in PK-15 cells.This assay provided a possible development of future prevention and control strategy of PRV.4.Expression analysis of interferon and pro-inflammatory cytokines in ISG15-over-expressing cellsThis study focus on determining the effect of porcine ISG15 on the expression of porcine interferon signaling.The recombinant expression vector(p CAGGS-HA-p ISG15)was successfully constructed,and transfected into PK-15 cells using LipofectamineTM2000.Under the condition of no stimulation or treatment with poly(I:C),the promoter activity of IFN-β was tested by dual-luciferase reporter system.The results revealed that overexpression of porcine ISG15 promoted the promoter activity of IFN-β.Subsequently,expression of IFN-β was tested by ELISA and the transcriptional level of IFN-β、OAS and IL-8 were dectected by q RT-PCR in ISG15-over-expressing cells and vector control cells before and post-JEV infection.The results revealed that STAT1-dependent expression of IFN-β、OAS and IL-8 in ISG15-over-expressing cells and PRV-infected ISG15-over-expressing cells were higher than those in vector control cells and PRV-infected vector control cells.This assay provided foundation for investigating the molecular mechanism of ISG15 against PRV in future.