UL27 Gene Of Duck Plague Virus Antigen Protein Domain Prokaryotic Expression, Purification, Antibody Preparation And Application

Base on duck plague virus(DPV) UL27 gene(GenBank accession number EF608147) to carry out the following series of studies:Bioinformatics Analysis of DPVUL27 and predicted epitope peptides.The main antigen domain cloning, prokaryotic expression,preparation of polyclonal antibody and Subcellular localization.Using Western blot and fluorescence quantitative PCR analysis time course of UL27gene expression and transcription.Prepare subunit vaccine using UL27-M proteinum and carry out experiment of infect protection.The results are as follows:1.Using DNA Star Protean software,the UL27 gene amino acid of the duck plauge virus(DPV) CHv strain(GenBank accession number EF608147) was analyzed on secondary structure and hydrophilicity and antigenic index,with the B cell epitopes to be predicted.The results showed that the epitopes of UL27 were situated in aa151-550,2.The genomic DNA of DEV CHv was extracted as PCR template,One pair of specific primers was designed by using primer5.0.A fragment coding for UL27 major antigen epitopes(451-1650bp) was amplified by PCR technique and was expressed by prokaryotic expression.Western-blot analysis indicated that multiclone anti-serum of DPV had specific reaction with the recombinant protein.The expression product was purified successfully 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 ELISA showed that the valence was up to 1:819200,has neutralize activity.3.Immunolocalization of DPV gB gene products in virus infected DEF Immunolocalization detection was observed using immunofluorescence technique, results shown that specific fluorescence appeared on nuclear membrane as early as 8hours post infection and a great deal of specific fluorescence concentrated in the cell nucleus by 12 hours,24h fluorescence maximum and transfer to the cytoplasm,36h fluorescent almost around cell membrane,48h fluorescent distribute all regions.4.Time course of gene products in DPV infected host cells and transcriptional analysis.Fluorescence quantitative PCR shows the DPV UL27 gene transcripts appeared as early as 6h post infection,then Fluorescence signal intensity increased steadily and reached a peak at 14 h,and remained detectable up to60 h,which owes the typical characterization of herpervirus late genes.A time course of DPV infected DEF were analyzed by western-blot using the polyclonal antibody IgG against DPV UL27M.A specific immunoreactive band migrating was observed at the expected position for protein DPV gB(about 110kDa) with maximal amounts between 60 and 72h.