Cloning, Expression And Polyclonal Antibody Preparation Of DPV US10 Gene

Through theoretical research and scientific experiments, we studied on molecular properties analysis, prokaryotic expression, polyclonal antibody preparation, intracellular localization, transcription dynamic analysis of DPV US10 gene (GenBank accession number:EU195084). The main results are as follows:1. Molecular analysis of DPV US10 gene and its encoding proteinThe whole gene length of the duck plague virus strain CHv US 10 gene was 969bp, and this gene encoded an estimated 322 putative protein, which contained the conserved domain of the Gene66 (IR5) protein. Subcellular localization revealed that the US 10 protein was mainly resided in cytoplasm. Post-translational modification analysis of the US10 protein indicated that 35 potential phosphorylation sites and five pairs of disulfide bond existed in the protein. There were no signal peptide and transmembrane region in this protein. Phylogenetic tree of the amino acids sequences showed this gene had a closer evolutionary relationship with Mardivirus. Codon usage bias in the US10 gene was relatively uniform, and there was no two or more consecutive rare codons existed in this gene.2. Prokaryotic expression and polyclonal antibody preparation of DPV US10 geneAccording to the nucleotide sequence of the duck plague virus US 10 gene, we designed a pair of primers by using Oligo 7.0 software for PCR amplification of the whole US 10 gene. The length of the amplified fragment was 988bp, then this PCR product was directly sent to TaKaRa company to construct the pMD18-T/US10 recombinant plasmid. Later, we constructed the pET32a(+)/US10 recombinant plasmid and expressed in E. coli BL21 host bacteria. The fusion protein was mainly existed in the form of inclusion body. Western blot analysis showed that the fusion protein reacted with rabbit anti-DPV serum, and indicated that there was the immuneoreactivity between the protein and rabbit anti-DPV serum. Through nickel chelating affinity chromatography, the fusion protein was purify and collected. Then, we used the purified protein and adjuvant to immune rabbits for preparing polyclonal antibody. The agar gel diffusion test showed that the highest antiserum titer reached 1:8.3. The intracellular localization of DPV US10 proteinIntracellular localization of DPV US 10 protein was.analysed by the indirect immunofluorescence. The results showed that US 10 protein was mainly appeared in cytoplasm. The specific fluorescences of US 10 protein were first observed at 12h post infection. And the fluorescence intensity was enhanced first and then weakened as time goes on.4. The transcription dynamic analysis of DPV US10 geneBy using the SYBR Green I real-time fluorescent quantitative PCR, we detected the different time points of transcripts of the US 10 gene after DPV infected its host cell DEF in vitro. The results showed that the US 10 gene was detected at 8h after infection, up to a peak at 72h, and then began to decrease. Drug inhibition assay indicated that the US 10 gene transcription was inhibited by ganciclovir and cycloheximide. In conclusion, the US 10 gene is a late gene.