A Preliminary Study Of Anti-Classical Swine Fever Virus Activity Of Porcine Ifn-a And Mx2 Protein

Classical Swine fever which caused by swine fever virus,is a highly potent and contagious disease.Clinically,acute swine fever caused by the strong poison,has higher morbidity and mortality rates and swine fever caused by attenuated infection does not show clinical signs so it may be difficult to detect.Classical swine fever is a worldwide distribution,once outbreak,the swine industry will cause significant economic losses,so the OIE included it to statutory Class A infectious diseases,our country also included it as a category of animal diseases.The genome of classical swine fever virus is a linear single-strand RNA,its full-length size is about 12.3kb.Its 5' end has no methylated cap structure,but a stable secondary structure,and there are existingmultiple invisible codons of AUG which do not start to translate.And its 3' end has no poly(A)tail structure.In all pestiviruses genus,the 3' end has high conservatism,particularly in the area of last about 100nt.IFN-a is able to exert its antiviral activity because it can associate with the type ?interferon receptors on the cell membrane.After internalized,IFN-a enters into cells to induce the synthesis of certain proteins with effector cell activity.Type ? interferon is the first line of cellular immunity to defense viral pathogens and the result of its signal transduction is to widely raise the levels of interferon-stimulated genes(ISGs)to check different stages of the viral life cycle,from the endocytosis,copy and release.Mx protein is innate,keyadjustment factor of cells against viral infections,whichtype ?interferons(?/?)and Type ? interferon(?)produce in the cells.They belong to a large superfamily of GTP dynamic protein,thefamily connectswith the intracellular vesicle transport and intracellular environment.For RNA viruses,Mx protein has a broad anti-viral activity,the virus was suppressed in the early stages of its life cycle,that is,after getting into the host cell and before the genome starting to replicate.Since being found to have antiviral activity,Mx protein began to be widely studied,moreover it has become the focus of anti-virus research.So far,researchers have found that human MxB protein inhibits the proliferation of HIV-1,human MxA protein and porcine Mx1 can inhibit the proliferation of CSFV,porcine Mx2 protein inhibits the proliferation of JEV and VSV.In this study,we use porcine Mx2 protein to develop the following experiment:1.Study on the effects on the anti-CSFV activity of IFN-aThis research adopted the method of real-time fluorescence quantitative PCR to detect the inhibition of CSFV by IFN-a.Here,we studied the inhibitory effects of IFN-a against classical swine fever virus(CSFV)in dose-dependent manner and time-dependent manner.The result showed that,cells treatedwith 10,50,100 and 300ng/mL concentrations of IFN-a,ortreated with the increasing-concentration IFN-a,CSFV was inhibited more obviously(P<0.01).Compared to the control group,the inhibition rates were 40%,67%,83%and 91%.Moreover,cells treated with IFN-a,the inhibitory effect on CSFV at 24 h,about 95%.In addition,the changes of Mx2 mRNA from cells treated with IFN-a were significantly increased(P<0.01).The results showed that Mx2 mRNA in cells treated with IFN-a was significantly increased(P<0.01),in a time-dependent manner within 24 h.2.Study on The anti-CSFV activity of porcine Mx2In this study,two pairs of specific primers were designed according to the sequence of porcine Mx2 gene.the first half(1170 bp)and full length(2136 bp)of the Mx2 gene were amplificated,then cloned into eukaryotic expression vector pFLAG(1170 bp)and pEGFP(2136 bp),the results suggested that two recombinant plasmids pFLAG-Mx2 and pEGFP-Mx2 were successfully constructed.Using Western blot analysis,we confirmed that two recombinant plasmids could be expressed in cells and then protein molecular weigh were 48 kDa and 102 kDa,respectively.After confocal microscope,the subcellular localization of Mx2 protein were in cytoplasm.Then,porcine Mx2 protein was detected the inhibition on classical swine fever virus.The recombinant plasmids pFLAG-Mx2 and pEGFP-Mx2 were transfected into PK-15 cells,the results of real-time PCR showed that pFLAG-Mx2 and pEGFP-Mx2 could inhibit the proliferation of CSFV and the inhibition efficiency were 56%and 67%.During 12-24 h,the role of inhibition gradually increased in a dose-dependent manner.The data of indirect immunofluorescence assay showed that number of green fluorescent spots in cells transfected with plasmid pFLAG-Mx2 were significantly less than that of cells transfected with plasmid pFLAG.It indicated that Mx2 protein from recombinant plasmid pFLAG-Mx2 had an inhibitory effects on CSFV.Also,the results of confocal microscopeshowed that Mx2 protein from plasmid pEGFP-Mx2 could inhibit the proliferation of CSFV.However,this inhibition was not finished thro?gh the interaction with the CSFV glycoprotein E2.3.The prokaryotic expression,antibody preparation and applicationof the classical swine fever virus Core proteinAccording to the gene sequence of classical swine fever virus capsid protein,we designed a pair of primers.With the method of RT-PCR,we got a complete core gene from C-strain.After Enzyme digestion and sequenced,we cloned it into the vector of pColdI carrying a His tag,then we built the plasmid pColdI-Core before it was transformed into E.coli Rosseta 2(R2).At last,we used IPTG to induce the expression of the protein and identified the effect of the expression.The results showed that:The Core gene expressed after being induced by the final concentration of O.lmmol/L IPTQ and the expression in total of 32.6%of the somatic protein,SDS-PAGE showed the relative molecular of target protein was mass of 20 kDa.Western blot illustrated that Core protein could specifically react with His monoclonal antibodies and classical swine fever virus-positive antiserum specific,there was a single reaction zone.After cutting glue,we immunized Balb/c mice to prepare specific antiserum.And Western blot showed that there was a clear specific band after the antiserum reacting with the capsid protein.Identified by confocal,antiserum could react with swine fever virus particles in the cytoplasm,which showed the success of Core gene expression,antiserum we had prepared possessed biological functions.