Cloning, Expression, Transcription Phase Analysis And Cellular Localization Of The UL33 Gene Of Duck Plague Virus

This article has carried out series researches on the newly discovered UL33 gene (assigned accession no.EF643556) of the duck plague virus(DPV) by the means of sequence characterization and bioinformatics analysis,cloning,prokaryotic expression, preparation of polyclonal antibody,time course of gene products in DPV infected host cells and expression phase analysis.Results were reported as following:1.Sequence characterization of the DPV UL33 geneThe DPV UL33 gene was composed of 408 nucleotides encoding for a polypeptide of 135 amino acid residues.Through the prediction and analysis of signal peptide, transmembrane domain,phosphorylation site and hydrophobicity,found UL33 had not signal peptide and transmembrane domain,only had eight phosphorylation sites. Cladogram analysis found UL33 had belong toα-Herpesvirus,which was relationship with MDV,ILTV,HVT.2.DPV UL30 gene clone,prokaryotic expression and polyclonal antibody preparationAn prokaryotic expressional plasmid pET-32/UL33 was constructed and a polypeptide with a size corresponding approximately to that expected for the recombinant protein (about 37kDa) were highly expressed with 0.2mM IPTG at 37℃and found in large amounts in crude cell extracts.the recombinant fusion protein were expressed under IPTG induction.Purified protein was used to immunize rabbit for the UL33 anti-serum preparation.High specific IgG polyclonal antibody was obtained by purification using the caprylic acid and ammonium sulfate precipitation and High-Q anion-exchange chromatography.3.Immunolocalization of DPV UL33 gene products in virus infected DEF and Transcription phase analysisFluorescence through the cell-mediated immunity to virus infection of the subcellular localization DEF testing,results showed that the one is the UL33 protein isolated in nucleus;the one is the virus in two hours after inoculation of duck embryo fibroblasts can be found in punctate green fluorescence.2h after the gradual increase,8hafter the green fluorescence of spots can be observed very clear and inoculation of the detected fluorescence to reach the largest number and around the nucleus has some distribution.Transcription phase analysis used fluorescent quantitation PCR to detect gene transcription information.The result shows that 1h after beginning to transcribe,8h reached the summit,and 32h decreased the minimal level.