The Prokaryotic Expression, Time Phases Of Transcription And Expression And Subcellular Localization Of DPV UL39Gene

Based on the information of DPV-CHv genomic library and the sequencing from our laboratory, we have analysised the molecular characteristic of DPV UL39gene. Specific primer was designed and synthesized according to the UL39gene sequences form DPV-CHv in order to amplification the target gene (GenBank Accession No. EU071042), prokaryotic expression and purification the UL39gene encoding protein, preparation of rabbit anti-UL39protein polyclonal antibody, we have analysised the transcription and expression phase, distribution of UL39gene expression products in the DPV infected DEF.1. Molecular characteristic analysis of UL39gene from DPV DPV UL39gene was composed of2433nucleotides encoding for a polypeptide of810amino acid residues. The protein has40potential phosphorylation sites and4N-glycosylation sites, without the signal peptide and the transmembrance region. Alignment the homologous from other20reference alphaherpesviruses, we have constructed the phylogenetic tree, proved that DPV R1protein had the closest evolutionary relationship with the Mardivirus genus of the Alphaherpesviruses.2. Prokaryotic expression and polyclonal antibody preparation of DPV R1Sent the PCR amplification products of UL39gene used high fidelity enzymes to TAKARA (DALIAN) to construct the recombinant plasmid pMD2O-T-UL39, then sequencing of cloned fragment. To digestion this recombinant plasmid with restriction enzymes (BamHl and Hindlll), recover purified target fragment product, and then orientational insert in the pET-32a(+) vector precut with the same enzymes to construct the recombinant plasmid pET32a-UL39. The recombinant plasmid of pET32a/UL39was transformed into Rosetta (DE3), IPTG was added to induce expression, R1mainly exist in the inclusion bodies, the molecular weight about110kDa. After optimizing prokaryotic expression conditions, we determined the optimum temperature, inducement time and concentration of IPTG was that at30℃for6h with IPTG of0.4mmol/L. The means we employed were cut the gel, and then electrodialysis, the purity of recombinant protein we have collect can meet the needs of immunize the rabbit. The protein had been emulsified with adjuvant, the rabbit was immunized with the emulsified protein, and agar diffusion reaction showed that the antibody titer was up to1:16～1:32. 3. Transcription and expression phase analysis of UL39gene in DPV infected DEF in vitro The detection result of the transcription phase of UL39gene in DPV infected DEF was analyzed by using FQ-PCR showed that UL39mRNA can be detected beginning of2-4h post-infection(p.i), and reached a peak at36h p.i, then reduced at48h p.i. The nucleic acid and protein inhibition tests were used to identify the gene types of DPV UL39gene. The analysis of expression phase of DPV UL39gene was confirmed by using Western-blot assay, the UL39protein expressed at a low level at early time of3-6h p.i, then reach to the peak level at36h p.i. Comprehensive analysis and judgement the results of above tests, we can deduce that DPV UL39gene was a Immediate-early gene.4. Localization analysis of DPV UL39gene products in DPV infected DEF in vitro The distribution of DPV UL39protein in DPV infected DEF was examined by indirect immunofluorescence assays in vitro, the results indicate that specific fluorescence mainly located around the cell nucleus (perinuclear).