Cloning, Prokaryotic Expression, Cell Localization And Transcription Phase Analysis Of Duck Plague Virus UL28 Gene

A pair of specific primer was designed and synthesized according to the duck plague virus UL28 gene based on Duck plague virus gene library of author's lab.The UL28 gene was PCR amplification(GenBank NO.EF643557)。Then,the molecular characteristics and the usage of codon choice of UL28 gene was analysised.And research work carried out the following:An oligonucleotide probe was designed according to the conserved domain of DPV UL28 for dot-blot hybridization with genomic to detection of Duck plague,prokaryotic expression,preparation of polyclonal antibody,time course of gene products in DPV infected host cells and transcriptional analysis.Results were reported as follows:1.Molecular characteristics analysis of the DPV UL28 gene DPV UL28 gene was composed of 2412 nucleotides encoding for a polypeptide of 803 amino acid residues.Its encoding protein(ICP18.5) had a conserved domain named Herpesvirus Processing and Transport Protein(PRTP).Moreover,the amino acid sequence of DPV UL28 gene had higher homology with UL28 homologous protein of Alphaherpesvirus than others.Phylogenetic tree analysis showed that on taxonomic status the DPV could be grouped in Alphaherpesvirus subfamily.that was a branch of Alphaherpesvirus subfamily of waterfowl.Codon usage analysis results show that codon usage of the larger differences in the choice for encoding the same amino acid, it showed strong preference.2.DPV UL28 gene prokaryotic expression and polyclonal antibody preparation The UL28 gene from PCR connected to the pMD18-T vector,then sequencing of cloned fragment with the correct orientation to insert in the pET-32a (+) vector.The UL28 gene was sub-cloned into prokaryotic vector pET-32a(+) were digested with BamHI and SalI.Then the recombinant plasmid pET32a/UL28 was transformed into E.coli BL21(DE3) strain and expressed under IPTG induction. SDS-PAGE analysis showed that the induced expressed protein was about 110KD. The proteins were highly expressed at 25℃for 5 h with IPTG of 0.1mmol/L,and found in large amounts in inclusion bodies.The expression product was purified by passing the Ni~+ affinity chromatograph collumn using the recombinant protein with a tag of 6×His.The rabbit was immunized with the purified protein,and agar diffusion reaction showed that the antibody titer was up to 1:16～1:32.3.Expression and cell localization of DPV UL28 protein in virus infected DEF Immunolocalization detection was observed using immunofluorescence technique,results shown that specific fluorescence appeared in cell nucleus as early as 2 hours post infection.With the extension of the infection time,the more specific fluorescence appeared in the nucleus,12h strongest fluorescence was detected,mainly in the nucleus.After the fluorescence intensity gradually weakened,the virus associated with the erosion of the cleavage nucleus,nuclear fluorescence gradually weakened diffusion.it difficult to detect fluorescence after 48h.4.Transcriptional analysis of UL28 gene in DPV infected host cells The transcription analysis of UL28 gene in DPV infected host cells was detected by FQ-PCR,The results showed that DPV UL28 gene transcription product of a sharp rise in first with the increase of infection time,and then a continued downward trend. As early as 0.5h post infection had detected the beginning of UL28 gene transcription, followed by rapid amplification of transcription products,and reached a peak after 12h post infection,the decline in the relative value down to a certain level 24h post infection,the detection of the relative value of close to zero 32h post infection.