| Interferons（IFN）,produced by animal body have diverse effects on innate and adaptive immune cells during infection with viruses,bacteria,parasites and fungi,directly or indirectly through the induction of other mediators.China is the biggest country of feeding ducks.However,virus diseases are major diseases that puzzled the duck industry.Currently,commercial vaccines only showing some promise in their strict specificity against particular pathogens.Using recombinant duck IFN is an efficacious method of maintaining duck virus diseases.In view of this,we cloned and expressed duck interferon-beta（duIFN-β）and carried out its antiviral activity to duck virus.The main contents include:1.Cloning and sequence analysis of Duck type Ⅰ interferon IFN-βThe total RNA as a template was extracted from duck liver,RT-PCR was perform using duIFN-βprimers,to amplify duIFN-βand gene fragment size is 729 bp.Amino acid sequence analisis showed that duIFN-βhad high homology with chicken IFN-β.Analysis of the duIFN-βamino acid sequence predicted that first the 29 amino acid residues at N-terminal encoded its signal peptide.2.The expression of Duck type Ⅰ interferon IFN-βin E.coliOn the basis of our successful cloning of type Ⅰ IFN in duck,we removed the signal peptide-coding sequence constructed a recombinant p ET-28a-tduIFN-β,and then tansformed into E.coli Rosetta（DE3）.We established a stable system of protein expression a purification of duck recombianant IFN（tduIFN-β）.As determined by SDS-PAGE,the tduIFN-βfusion protein is about 26KDa.Highly purified and bioactive tduIFN-βwas harvested by affinity chromatography by using Ni2+-NTA His-Bind Resin.The protein possessed relatively low cytotoxicity on DEFs determined by MTT assay,and DEF（duck embryo fibroblast）/VSV（vesicular stomatitia virus）micro-cytopathic effect inhibition method was used for the determination of purified tduIFN-β.The bioactivity of tduIFN-βprotein was 2.0×104 U/mg.3.Duck type Ⅰ interferon IFN-βinhibits Duck Virus DHAV,DTMUV and DRVIn order to study the effect of duIFN-βon the proliferation of duck virus,DEF Cells were pretrested with tduIFN-βprokaryotic expression products,then infected with DHAV,DTMUV and DRV,respectively.TCID50 were performedto detect the proliferation of the viruses.The results showed that duIFN-βcould inhibit the proliferation of duck viruses,which has broad-spectrum.Animal experiments showed that recombinant tduIFN-βcould resist the DHAV infection.4.Duck type Ⅰ interferon duIFN-βinduce ISGs expression patternAdditionally,the effect of duIFN-βon the expression of duck interferon-stimulated genes（ISGs）was analyzed by real-time fluorescence quantitative PCR.We observed that several IFN-stimulated genes（ISGs）were greatly up-regulated on both DEF cells and ducks. |
