Cloning, Expression And Function Studies Of The UL34 Gene In The Replication Procedure Of Duck Plague Virus

1.Cloning and molecular characteristic of the DPV UL34 gene.A 831-bp complete open reading frame(ORF) of the duck plague virus UL34 gene(GenBank accession no,EF643562) was isolated in our laboratory by constructing the genomic library of DPV CHv strain.This gene was cloned and sequened.A large number of bioinformatics software was then used to analyze its molecular characteristics.The results indicated that this gene encodes an estimated 276 putative protein,contains the conserved domain of the Herpesvirus UL34-like protein.Phylogenetic tree based on the amino acids sequences showed DPV has a close evolutionary relationship with GaHV-2,GaHV-3, MeHV-1,EHV1,CeHV-9 and HHV3,which classified into the Mardivirus or Varicellovirus,indicating that the DPV should be placed into a single cluster within the subfamily Alphaherpesvirinae.An obvious transmembrane region was located between 252-274 amino acids and also has a microbody targeting signal in the C terminal,but without a signal peptide.Four Casein kinaseⅡphosphorylation sites,four Protein kinase C phosphorylation sites,five N-myristoylation sites and one N-glycosylation site were searched in the protein.Most of the total proteins probably situated in the nucleus.The codon usage patterns of the UL34 gene in DPV showed it was a low expression gene with AT-riched at the third position.These results strongly suggest that the UL34 protein of DPV-CHv strain was a tail-anchored typeⅡmembrane protein,and the expression in yeast maybe the better,as well as provide rational and valuable data to elucidate biological function of the gene.2.Expression of the DPV UL34 protein and antibody preparation.The recombinant prokaryotic expression vector pET-32/UL34 was constructed and induced by IPTG and a size of 52kDa protein was obtained,which was conincindence with the expected result.Soluble analysis indicated that the recombinant protein was exsited in a form of inclusion body.The optimal concentration of the IPTG was 0.4mmol/L and the induction time was 4h at 37℃.The titer of the antibody prepared with the anti-UL34 recombiant protein against rabbit dected byenzyme linked immunosorbent assay was 1:153600.Western blot results according to the recombianant UL34 bacterium as antigen showd that it can reacte with the complete serum of DPV,as well as reacted with prepared anti-UL34 serum.These results proved that the UL34 encoded protein can stimulate anti-DPV immunological reaction and the purified IgG of anti-UL34 serum involved with ammonium sulfate salting-out and anion-exchange chromatography has a advantage of high performance and strong specificity. 3.Cellular location of the UL34 protein in DPV-infected DEF.Purified anti-rabbit-UL34 serum was used as the primary antibody in IFA to monitor the cellular location of the UL34 protein in DEF cells.The result indicated that the encoded protein assembled in the nuclear membrane of the infected cells,the specific fluorescence was first detected at 12h post-infection.At 24h post-infection,a large number of green fluorescence was concentrated in the nuclear membrane.After 48h post-infection,with the disintegration and morphological disruption of the cell,the strength of the specific fluorescence was widely depressed.These experimental results further proved that the UL34 protein was a typeⅡmembrane protein,which was partly coincidenc with the bioinfomatics analysis, and the UL34 protein was likely play an impotant role in the neucleocapsid budding of the DPV.4.Distribution of the UL34 antigen in the DPV-infected ducks.The adult ducks was artificially infected with DPV-CHv,different sample tissues from duks at 4-day postinfection were obtained and then embedded in paraffin.Paraffin sections were cut from each tissue,mounted,and baked to involve with immunofluorescent staining.The strong positive immunofluorescence was found in the liver,spleen,bursa of Fabricius, glandular stomach,duodenal,jejunum,ileum,rectum,cecum and the lung of 4d post-infected ducks,while showed less immunofluorescence in brain,musule,Harder's glands.These data strongly suggest that the immunological and digestive organs are likely the target organs in DPV infected ducks,and the establishment of IFA on the basis of UL34 protein as the primary antibody overcome the difficulty of virus purification,which can served as a sensitive,visible and rapid diagnostic method.5.Transcription and expression phase analysis of the DPV UL34 gene.We generated a pair of primers U3/U4(amplification product of 93bp) according to the UL34 gene sequence.RNA from DEF cells at different infection times including1h,2h,4h, 8h,12h,16h,18h,24h,28h,32h,36h,42h,48h,52h,60h,66h,72h was extracted by Trizol and performed reverse transcription into cDNA.Fluorescent quantitation PCR amplification showed significant increase at 12h,appearing a peak at 28h;and then with sharp decrease until 32h to achieve comparatively stable changes.The purified UL34 antibody was reacted with the viral protein of DPV substracted by freezed thawing several times and then performed Western blot test.The results showed that the UL34 protein was first detected at 12h,followed with gradually increasing.These findings proved that the transcription and expression phase of the DPV UL34 gene was conincidence with the virus propagation,and this gene was a late gene with a characteristic of transcript and expression at the same time,further supported the conclusion that the UL34 gene was necessary to the viral replication,and play an important role in the primary envelopment and keep the complete virulence of the DPV.