The Preliminary Study On The Interaction Between Duck Plague Virus UL1 Protein And GH

1. Bioinformatic analysis of UL1 gene of Duck Plague Virus. DPV UL1 gene has 711 nucleotides encoded a protein of 236 amino acids. DPV gL protein has a molecular weight of 26221.08Daltons. The contents of Val, Gly, Arg were 9.8%,8.5%,8.1%, respectively. The phylogenetic tree shows that DPV gL protein has close evolutionary relationship with the genus Simplexvirus. The online analysis of the physicochemical properties demonstrates that the protein has ten potential phosphorylation sites and five potential O-linked glycosylation sites, and without both the signal peptide and the transmembrance region. The secondary structure results reveal that random coil dominate among secondary structure elements followed by alpha helix, extended strand and β-turn for all sequences. In addition, the subcellular localization of gL protein largely locates at the cytoplasm and mitochondria with a probability of 17.4% and 47.8%. All the data will help a basis for further functional and physiological features study of the DPV gL protein.2. Cloning, prokaryotic expression, purification and polyclonal antibody preparation of DPV ULl truncated gene. The full-length DPV UL1 gene can’t express in prokaryotic expression system, so we amplified the DPV UL1 truncated gene(gLt) which don’t contain the signal peptide. Cloning the UL1 truncated gene into the pMD19-T vector, then targeted inserte the gLt fragment into pET-32a(+) and transform it into expression host E. coli. Next, we optimiz the expression conditions, for instance, inducement time and concentration of IPTG. Finally, we found the recombinant protein could express in high efficiency in the BL21(DE3), at 37℃, induced 8h by 0.8mmol/L IPTG. Using the purified gLt recombinant protein to immunize rabbits and collected the serum when its titer was up to 1:32. In the end, we extracted and purified the IgG of the serum by using ammonium sulfate precipitation and ion-exchange column chromatography respectively.3. Subcellular localization of protein encoded by DPV UL1 gene in DPV infected DEF. Using the indirect immunofluorescence analysis to examine the intracellular localization and distribution of protein encoded by DPV UL1 gene.The specific fluorescences of DPV UL1 protein were observed only in cytoplasm, and they were first observed in the DPV infected DEF at 7h p.i, then the specific fluorescences clustered strongly and slowly, the intensity of fluorescences achieved the maximum at 46h p.i. The results suggested that DPV ULl was a late gene and provided the foundation for function research of DPV UL1 protein.4. Gene type identification and expression kinetics analysis of DPV UL1 gene. We studied on the DPV UL1 gene transcription with the antiviral drugs, use CHX, GCV to identify the gene types of UL1 gene, the result showed that UL1 gene is a late gene of DPV. At the same time, the western-blot assay of DPV UL1 expression kinetics showed that the relative expression level of DPV UL1 gene was at a low level in the first 8h p.i, and its expression level to the maximum at 24h p.i, and thereafter both of them declined.5. Indirect immunofluorescence analyze the intracellular co-localization of gL and gH of DPV. We constructed a eukaryotic recombinant plasmid pCMV-Myc-gL. Then transfected the recombinant plasmids pCMV-Myc-gL and pCMV-HA-gH to the HEK293 cell by liposome Lipofectamine 2000, and analyzed the intracellular co-localization of gL and gH by indirect immunofluorescence test (IFA). The result suggested that DPV gL possibly can interact with gH.