Expression Of ?NS Nonstructural Gene Of Muscovy Duck Reovirus And Preparation Of Polyclonal Antibody

Muscovy duck reovirus(MDRV)infects 4 to 45-day-old Muscovy ducklings with a high mortality ranging from 10%to 50%.This acute infectious disease is mainly characterized by clinical symptoms of soft foot,diarrhea,and lesions with liver and spleen swollen and covered with acicular white necrotic foci,and Muscovy ducklings resistant to MDRV always display significantly stunted growth.Besides,other pathogens secondary to MDRV infection often aggravate the disease due to suppression of immune function of sick ducklings.In the last few years,MDRV infection has become one of the major infectious diseases in Muscovy ducks production.Chicken reovirus(CRV)?NS protein,a nonstructural RNA-binding protein,possesses functions of accumulating in viral factories of CRV-infected cells,and may play an important role in viral RNA assembly and replication.Presently,no functional studies on MDRV ?NS have been reported,alignments of MDRV YB strain ?NS gene segment and its deduced amino acid sequence implied that MDRV ?NS protein might have similar functions with CRV vNS protein.Expression of ?NS nonstructural gene of MDRV and preparation of polyclonal antibody were performed in this study,which will be helpful for further functional studies and acquirement of genome replication mechanism and pathogenesis of MDRV,and for prevention and control of MDRV infection,eventually.To express and prepare polyclonal antibody against ?NS protein of MDRV-YB strain,the ?NS gene was amplified and inserted into prokaryotic expression plasmid pET-32a,and then expressed in E.coli BL21(DE3)with IPTG induction.As expected,SDS-PAGE analysis showed robust expression of a 58 ku fusion protein(termed p-?NS)in both soluble and insoluble fractions.In addition,the IPTG induction time,concentration and temperature have been optimized to be 4 h,1 mmol/L and 37?,respectively.Then the recombinant proteins in both soluble and insoluble fractions were purified to homogeneity using Ni2+-affinity chromatography and could be recognized by the MDRV positive serum in immunoblot assays,which indicated that the expressed fusion protein had high reactogenicity.Finally polyclonal antibody was prepared from the rabbit immunized with the purified soluble p-?NS protein,and the ELISA titer could be greater than 1:32 000,Western-blot analysis further confirmed that the polyclonal antibody could react specifically with p-?NS and MDRV-?NS protein,which would be positively useful in further functional studies.To express e-?NS protein of MDRV-YB strain in eukaryotic cells,the ?NS gene was amplified and correctly cloned into eukaryotic expression plasmid pCI-neo-flag,and then transfected in DF-1 cells with ViaFect transfection reagent,and transcription and expression levels of ?NS gene were measured by semi-quantitative RT-PCR and Western-blot,respectively.Results showed that the ?NS mRNA levels increased gradually at 0?36 h post-transfection,while with a declination at 48 h;Western-blot also confirmed that the expressed e-?NS protein could react specifically with both anti-flag mouse monoclonal antibody and anti-p-?NS rabbit polyclonal antibody,and the maximal expression level of e-?NS protein was achieved at 36?48 h post-transfection.All above results indicated that MDRV-YB aNS gene was successfully expressed in DF-1 cells,which would lay foundations for further functional studies.