The Prokaryotic Expression, Polyclonal Antibody Preparation And Its Application Of Duck Plague Virus UL7Gene

Duck plague virus (DPV) is a member of the Alphaherpesvirinae, who can cause an actue and often lethal infection disease of ducks, geese, swans and many other species of waterfowl within the order Anseriformes. DPV results in substantial economic losses in ducks industry. In this study, we study on bioinformatics analysis, gene cloning and prokaryotic expression, protein purification and preparation of polyclonal antibody, transcription time phase, kinetic class, intracellular localization and construction eukaryotic expression vector of DPV UL7gene (GenBank accession no. FJ222445). The results provide a reference for further study on DPV.1. Bioinformatics analysis of DPV UL7gene DPV UL7gene and its coding protein functions were analyzed by the bioinformatics software. The results indicated that DPV UL7gene contains a conservative domain. The coding protein had no signal peptide and transmembrane domain,12potential phosphorylation sites, a glycosylation site and a potential palmitoylation site. Subcellular localization prediction showed that UL7protein mainly located in the cytoplasm. In addition, the results of phylogenetic tree showed that DPV UL7gene was closed to UL7gene of Mardivirus from the Alphaherpesvirinae.2. Cloning, prokaryotic expression and antibody preparation of DPV UL7gene We designed for a specific primer according to DPV UL7gene sequence by the Oligo6.71software, the PCR amplification out whole ORF of UL7gene. Then the amplified fragment of DPV UL7gene was inserted into pMD20-T vector, after that the UL7gene was ligated with pET-32a(+) in order to constructthe recombinant plasmid pET32a-UL7, and it was transformed into express host bacterium BL21pLysS, induction expression with1PTG obtain the size of about50kDa fusion protein by SDS-PAGE analysis. Western blot analysis showed that fusion protein was specificity reaction with rabbit anti-DPV polyclonal antibody. The fusion protein was existence with inclusion body, and the expression optimal condition was0.5mmol/L IPTG at30℃for7h. The rabbits were immunized with the purified DPV UL7fusion protein. Furthermore, the agar diffusion test showed that the polyclonal antibody titer was up to1:32.3. Transcription time phase and kinetic class analysis of DPV UL7gene In this research, we employed the real-time fluorescence quantitative PCR method (qRT-PCR) to analyze transcription time phase of DPV UL7genes in duck embryo fibroblast (DEF). The results showed that the UL7gene transcription was detected as early as4h after infection, up to a peak at56h, and then the relative content began to decline. The results of UL7 gene transcription by qRT-PCR method indicated that the method is rapid, stable and good specificity. At the same time, we studied on UL7gene transcription with the antiviral drugs, the result showed that UL7gene belongs to a late gene of DPV.4. Intracellular localization of DPV UL7gene Intracellular localization and distribution of DPV UL7gene was used the indirect immunofluorescence analysis. We obtained the optimal conditions of UL7protein after optimization condition. The results showed that the specific fluorescences of DPV UL7protein were mainly appeared in cytoplasm and similar as bioinformatics analysis results. The UL7protein was first observed in bright fluorescent granules in the cytoplasm of infected cells at8h post infection (h p.i) and clustered strongly in the perinuclear region at48h p.i. The results provided the foundation for function research of DPV UL7protein.5. Construction and expression analysis of a eukaryotic recombinant plasmid encoding duck plague virus UL7gene To analysis DPV UL7gene expression in DF-1cell, we applied pMD20-T-UL7plasmid, then UL7gene cloning to eukaryotic expression vector pcDNA3.1(+), constructing a eukaryotic recombinant plasmid pcDNA-UL7. Recombinant plasmid was transfected DF-1cell by liposome LipofectamineTM2000, and then analyzed by RT-PCR, indirect immunofluorescence test (IFA) and Western blot of DPV UL7gene in DF-1. Results showed that the recombinant plasmid pcDNA-UL7expressed proteins located mainly in the cytoplasm in DF-1cell, and the molecular weight was about36kDa. All these results provided a reference to establish a stable expression DF-1cell line of DPV UL7protein.