| Known as duck plague virus,duck enteritis virus(DPV)could infect reaching up to 100%in birds such as ducks,geese,and wild waterfowls in the order Anseriformes,which could cause an acute infectious disease with a very high mortality.The DPV genome is double-stranded and linear DNA,consisting of a unique long(UL)region and a unique short(US)region.It encodes 78 predicted open reading frames(ORFs),and 65 ORFs are located in the UL region,including UL14 gene.There are 39 bp of UL14 overlapping with UL13.There were less information about DPV UL14 protein.Thus the bioinformatics analysis,prokaryotic expression and antibody preparation,transcription phase,expression phase,subcellular localization,the interaction with VP 16,and the deletion of UL14 genewere analysed,with the results as below:1 Bioinformatics analysis and characteristics of the DPV UL14 geneBioinformatics analysis was performed to predict the characteristics of the UL14 protein.The results revealing that UL14 gene encodes 154 amino acids(aa),including 29 acidic amino acids and 26 basic amino acids,hydrophobic 63 amino acids.Its molecular mass is 17.5 kDa,isoelectric point(IP)is 5.71,with molecular formula C760H1256N228O238S4.The UL14 protein may not be a secreted protein for having no signal peptide site.In addition,the analysis of the physico-chemical properties demonstrates that UL14 has 6 main antigenic determinants and 36 phosphorylation sites.There was no transmembrane region,and the antigen epitope were mainly concentrated in 18-23 aa,37 aa,49-50 aa,80 aa,105-113 aa and 127-150aa.Its second structure was confomed by 75.97%a-helix(HH),2.6%extended chain(EE)and 21.43%irregular curl(CC).Its third structure could not be predicted by the current database.2 Identification the tegument protein UL14 in DPVThe PCR product with the length of 664 bp containing UL14 gene,was coloned into pMD18-T vector.The recombinant vector of pET32a(+)-UL14 was constructed by sub-coloning.Then the recombinant vector was translated into E.coli BL21.After optimization of conditions,the 38.5 kDa protein(with 21 kDa tag of 6×His)was obtained by 0.2 mM IPTG 37?,by induced 6 h.And this protein could interact with antiserum of rabbit anti DPV.The UL14 recombinant protein was purified with nickel column chromatography.After immuning rabbit and mouse,the antiserum was harvest,with the 1:8 and 1:32 antibody titer.3 Characterization of the tegument protein UL14 in DPV(1)In order to detect the transcription of UL14 gene in proliferation after viral infection,we extracted the RNA at different time points(Oh,10h,12h,24h,36h,48h,60h,72h,84h)for the relative quantitative analysis.(2)Then we collected the infected cell at different time(Oh,10h,12h,24h,36h,48h,60h,72h,84h)to detect the expression of UL14 protein by western-blot analysis.(3)We analyzed the localization of UL14 protein in the host cell by indirect immunofluorescence method at different time points(12h,24h,36h,48h,60h,72h).(1)The results showed that the transcription of UL14 gene started from 24h post infected,and reached maximum at 72h.(2)In western-blot analysis the UL14 protein band appeared at 24h post infection,and the bands were obvious from 36h-60h,at 72h the band were larger than the other time point.(3)In the indirect immunofluorescence assay(IFA)assay,we could the green fluorescence in the cytoplasm at 36h post infected.At 48-60h post infected,the fluorescence increased and most distributed closed into nucleus.At 72h post infected,the fluorescence mainly distributed in the nucleus,while still a little distributed in the cytoplasm.4 Identification of the NLS region of DPV UL14 and its interaction with VP16The UL14 protein had no classical nuclear localization signal(NLS)analyzed by the bioinformatic analysis.While it contained four ?-helix(4-33aa?41-97aa?100-126aa and 146-152aa).Our previous data showed that the localization was mainly in nuclear.To find the sequece contained the NLS and the interaction with VP 16,these following tests were carried out:(1)Bioinformatics analysis of UL14 protein showed that UL14 protein contained 4 alpha-helixes.Based on this,we designed the truncated mutants.(2)The co-expression of pUL14 and VP16 was detected in transfected DEFs.A bimolecular fluorescence complementation(BiFC)assay was used to confirm a direct interaction between pUL14 and VP16.The results showed:(1)1-98 aa at the N-terminus of pUL14 played a role of the nuclear localization signal(NLS)region;(2)1-98 aa of pUL14 promoted translocation of VP 16 into nucleus to complete the virus life cycle.5 Deletion of UL14 inhibit replication and maturation of DPVWe constructed and characterized the UL14 deleted and revertant mutant viruses,based on the bacterial artificial chromosome recombination duck plague virus rescue system constructed by our laboratory.Then the mutants were confirmed by three methods,including the map of restriction digestion,western blot and immunofluorescence assay.The mutant viruses were characterized by plaque assay,virus reproduction curves and transmission electron microscopy(TEM)assay.Here,we demonstrated that knockout of UL14 resulted in an over 100-fold reduction in viral titer and a gross decrease in genome copies.The results of TEM assay revealed that the virion was irregular and the electron density of envelope was low in the absence of UL14.Taken together,our findings suggest the deletion of pUL14 inhibit DNA replication of virus and the integrity of envelope. |
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