Innate Immunity In DEF Cells Induced By Novel Duck Reovirus

Objective:Novel duck Reovirus(NDRV)is the pathogen of the newly emerging disease designated duckling hemorrhagenic and necrotic hepatitis since 2005 and causes much economic loss to the Chinese duck industry.The disease is characterized by multiple organs' hemorrhage and necrosis involving the liver,spleen and kidney.The host'innate immunity is the first line of defense against virual invasion.Host immediately innate immune responses which activated by virus infection can directly inhibit virus infection and promote host's acquired-immune response.In order to control NDR V infection,a preliminary study on innate immune response in duck embryo fibroblast cells(DEF)triggered by NDRV was carried out.The mule duck-origin IFN-?,IFN-?' cloning and IFN-?' prokaryotic expression Were also carried out.These data laied a foundation for further research on biological activity and clinical application of IFN-beta and IFN-lambda.Methods:Experiment I:DEFs were inoculated with NDRV and harvested at indicated time.Then DEFs' total RNA was extracted and reverse transcripted.The mRNA expression level of the innate immune related genes were analyzed by real-time PCR.Experiment II:Primers specific to duck IFN-? and IFN-? were designed according to GenBank accession number KM035791 and KJ206897.Duck IFN-p and IFN-?were amplified from DEFs using PCR method.Recombinant expression plasmids was constructed with cloned gene and carrier pET-3 0a digested with BamH I and Hind III,then transform to E.coli DH5a.The positive plasmids was confirmed by PCR,double enzyme digestion and sequence analysis.The recombinant plasmid was successfully constructed.Experiment ?:Constructed recombinant expression plasmids pET-DuIFN-? were transformed into E.coli BL21(DE3).The single colony selected was inoculated in liquid LB with kanamycin and induced expression with adding IPTG.The positive bacteria was collected and broken by ultrasonic in the ice.The soluble products and inclusion bodies of E.coli BL21 cells contained pET-DuIFN-? were analyzed by SDS-PAGE and Western blot.Results:Experiment I:The mRNA level of IFN-a,IFN-? and IFN-X was up-regulated rapidly in DEFs after infected with NDRV.The expression reached to their maximum at 48 hours post infection and started to decline gradually.The expression of pattern recognition receptors of MDA5,RIGI and TLR7 and antiviral protein ISG(interferon stimulated gene)of ISG12 and IFITM3 also showed similar pattern as interferons,but Mx1 was strong up-regulated from 0-60 h post infection.Experiment ?:Mule duck IFN-? and IFN-? with completed open reading frame were 729 bp and 558 bp in length,encoded 242 and 185 amino acids,respectively.Nucleotide homology analysis showed that the homology of mule duck IFN-? gene compared with duck IFN-a gene,duck IFN-? gene,goose IFN-? gene and chicken IFN-? gene was 87.2%?89.4%,100.0%,90.8%and 70.0%,respectively.The homology of mule duck IFN-? gene compared with chicken IFN-X gene and duck IFN-? gene was 75.8%?76.0%and 97.1%,respectively.Phylogenetic analysis indicated that mule duck IFN-? gene and anas IFN-? gene belong to the same evolutionary branches,mule duck IFN-? gene and gallus IFN-? gene was belong to independent branch.Mule duck IFN-X gene and anas IFN-? gene belong to the same evolutionary branches.Mule duck IFN-X gene and gallus IFN-? was independent branch.Experiment ?:Prokaryotic expression of mule duck IFN-? gene was carried out.Western blot analysis showed that the recombinant protein was 38 kDa while His tag protein control was 12 kDa.The fusion protein expression of mule duck IFN-? gene was successful obtained and existed as Inclusion bodies in E.coli.Conclusion:1.NDRV infection rapidly triggers host innate immune signaling with up-regulation of type I and type III IFNs and some critical ISGs in mule duck embryo fibroblasts.2.Mule duck-origin IFN-P gene and IFN-? gene was successful cloned and identified.3.The fusion protein of mule duck IFN-P was successful obtained in the recombinant plasmid designed as pET-DuIFN-?.