Cloning, Prokaryotic Expression, Transcription Phase Analysis And Subcellular Localization Of Duck Plague Virus UL41 Gene

According to the duck plague virus CHv strain UL41 gene sequence (GenBank accession No. EU071040) which was identified in our laboratory, we designed a pair of primers, then PCR amplified UL41 gene, did some bioinformatic analysis on the UL41 gene molecular characteristics and usage of rare codons, we also did the prokaryotic expression, protein purification and antibody preparation of DPV UL41 gene, the analysis of UL41 gene transcription phase and UL41 protein subcellular localization, by which obtained the following results:1. The molecular characterization analysis of DPV UL41 geneThe DPV UL41 gene open reading frame (ORF) was 1497bp in length, encoding a polypeptide with 498 amino acids (aa). Neither signal peptide nor transmembrane region was found through the online analysis. There are 24 potential phosphorylation sites when the threshold is above 0.5, including serine 9, threonine 8, tyrosine 7. Secondary structure analysis showed the random coil (C) and the alpha helix (H) occupied a large proportion.Tertiary structure prediction revealed it had homology with Flap endonuclease-1 (FEN-1). In addition, phylogenetic analysis revealed that the gene belongs to the Marek herpes virus, evolutionary relationship close to MeHV-1, GaHV-2 and GaHV-3.2. UL41 gene cloning, prokaryotic expression, protein purification and antibody preparationPCR amplified a DNA fragment with 1526bp by the designed UL41 primers, which contains the complete UL41 gene. Then cloned this fragment into pMD19-T vetor, constructed the recombinant plasmid pMD19-T-UL41. The plasmid pMD19-T-UL41 and pET-32a (+) were both digested with EcoR I and Xho I, then connected the target fragments to construct the recombinant plasmid pET-32a (+)-UL41. After being confirmed by PCR, both EcoR I and Xho I restriction endonuelease digestion and DNA sequencing, then pET-32a (+)-UL41 was transformed into E.coli BL21 (DE3) comperent cells for expression. DPV UL41 gene was successfully expressed after IPTG induced. The recombinant fusion protein was about 76KDa, in accordance with the expected size, which existed mainly in inclusion body. The optimum condition of expression was add to 0.2mmol/L IPTG at 30℃ for 10h. Western-blot detection showed that the recombinant protein could be recognized by rabbit anti-DPV serum. The recombinant protein was purified for preparation of rabbit anti-UL41 polyclonal antibody, agar diffusion tests showed that the titer of the serum was 1:8. The serum and UL41 recombinant protein had a specific recognition by Western-blot.3. The transcription phase of DPV UL41 geneFluorescence quantitative PCR detected the transcription of UL41 gene after DPV infected duck embryo fibroblast. Samples of different time were detected by SYBR Green I through relative quantitative method. The results showed UL41 gene began to transcription at 6h post infection, reached its peak at 36h, still had transcripts at 54h.4. Subcellular localization of DPV UL41 proteinThe subcellular localization of UL41 protein was detected by indirect immunofluorescence, the results showed the protein mainly located in cytoplasm. The fluorescence can be detected as early as 6h post infection. The fluorescence increases over time, fluorescence can still be detected at 48h post infection.