Co-localization Of UL17 Protein And UL25 Protein, UL26.5 Protein From Duck Plague Virus In Infected Cells

Little studies on DPV UL17 gene have been reported. This essay focused on the sequence characteristics, transcription and expression dynamic analysis, subcellular localization of DPV UL17 gene and UL17 protein. In addition, Co-localization of UL17 protein and UL25 protein, UL26.5 protein from duck plague virus CHV strain in infected cells. The results are as follows:1. Bioinformation analysis of DPV UL17 gene. The DPV UL17 gene was analysed by bioinformatics. The results indicated that UL17 gene encoded protein is 75.94298 kDa and comprising 684 amino acids. It had no signal peptide and the transmembrane region. At the same time the UL17 protein contained 19 phosphorylation sites.4 glycosylation sites and multiple antigenic epitopes. It was predicted located in nuclear and cytoplasm of infected cells.2. Prokaryotic expression, purification, polyclonal antibody preparation of DPV UL17 gene. Based on the UL17 gene sequence, a pair of specific primers was designed by Primer Premier 5.0. The amplified DPV UL17 gene was cloned into the pMD20-T vector. pMD20-UL17 and prokaryotic expression vector pET-32a(+) were digested with Xho I å'Œ BamH I. Then the UL17 gene was inserted into the XhoI å'Œ BamHI site of pET-32a(+). To express the UL17 protein, the recombinant plasmid pET32a-UL17 was transformed into BL21(DE3). SDS-PAGE result showed that the expressed protein is about 96 kDa The recombinant protein was purified with Ni-NTA method, to immunize BALB/C mice and rabbits for polyclonal antibody preparation. The highest antibodies titers were 1:4 and 1:32.3. Transcription and expression dynamic analysis of DPV UL17 gene. Based on the UL17 gene sequence, a pair of primers for FQ-PCR was designed. The result of drug inhibition study shows that DPV UL17 gene is a late gene. The real-time fluorescent quantitation PCR results shows that the UL17gene transcipt is firstly detected at 12 hour post infectionem, and reaches the highest at 48 hour post infectionem, then falls sharply at 72 hour post infectionem. Through Western Blotting the UL17 protein is firstly detected at 36 hour post infectionem in infected cells. Above all, DPV UL17 gene is a late gene.4. Subcellular location of DPV UL17 protein. The subcellular location of DPV UL17 protein in infected cells was detected by indirect immunofluorescence. The results indicate that UL17 protein locates in the cytoplasm firstly, then locates in nucleur and cytoplasm.5. Co-localization of DPV UL17 protein and UL25 protein, UL17 protein and UL26.5 protein in infected cells. The co-localization of DPV UL17 protein and UL25 protein, UL17 protein and UL26.5 protein in infected cells were studied by double indirect immunofluorescence. The results show that UL17 protein and UL25 protein co-locate in nucleur and cytoplasm. So do UL17 protein and UL26.5 protein.