DPV UL25 Gene Prokaryotic Expression And The Study Of Its Cellular Localization In Virus-infected Cells

This article has carried out series researches on the identified UL25 gene (assigned accession No EF643563) of the duck plague virus (DPV) by the means of sequence characterization and codon usage bias analysis, cloning, prokaryotic expression, preparation of polyclonal antibody and transcriptional analysis in DPV infected host cells. Results were reported as following:1. Sequence characterization and codon usage bias analysis of the DPV UL25 gene A 1797 bp complete open reading frame (ORF) of the duck plauge virus (DPV) CHv strain was definited in our laboratory. To identify the features of DPV CHv UL25 gene, a large number of bioinformatics analysis tools were used. The DPV CHv UL25 gene was composed of 1797 nucleotides encoding for a polypeptide of 598 amino acid residues. The polypeptide chain has 6 major hydrophobic domain, which are located in the 60-110 amino acid sequence spaces, 165-298, 325-435, 460-550-bit and bit 575-595; Secondary structure of polypeptide chain is characterized by the corner of (Turn) accounted for a larger proportion of the (content of up to 54%), folded (Sheet) contains 23% helix (Helix) content of 14%, and curly (Coil) content 9%; the protein located in the nucleus for nuclear targeting of proteins. Sequence comparison of UL25 among DPV CHv and 35 other reference herpesvirus strains showed that DPV CHv was more homologous to alphaherpesviruses than to betaherpesviruses or gammaherpesviruses. In a word, these experimental results provide valuable reference materials for clarifying the correlation function of UL25 gene.2. Characterization of codon usage bias in the newly indentified DPV UL25 gene A comparative analysis of the codon usage bias in the newly discovered UL25 gene of the duck enteritis virus (DPV) and the UL25 gene of 34 reference herpesviruses was performed. All of them was analysised for efective numbers codon(ENc), GC content and GC3s content respectively using the online-sofwares: CHIPS and CUSP. The codon adaptation index (CAI), effective number of codons (ENC), and GC3S values indicated synonymous codon usage bias in the UL25 gene of herpesviruses, and this synonymous bias was correlated with host evolution. The codon usage patterns of the DPV UL25 gene were phylogentically conserved and similar to that of the UL25 genes of the avian alphaherpesvirus. Although codon usage in each microorganism is different, there are no strain-specific differences among them. A strong bias towards A and T at the third codon position in the predicted polypeptide. Comparison of the codon usage in the UL25 gene of different organisms reveals that Eukaryotic Expression Vector system should be selected for UL25 gene expression. Analysis of variance (ANOVA) showed significant differences between the DPV and yeast UL25 genes (r = 0.04372, P < 0.01). The extent of codon usage bias in the DPV UL25 gene was highly correlated with the gene expression level, therefore the results may provide useful information for gene classification and furtherfunctional studies.3. DPV UL25 gene clone, expression and polyclonal antibody preparation An prokaryotic expressional plasmid pET32-UL25 was constructed and a polypeptide with a size corresponding approximately to that expected for the recombinant protein (about 80.0kDa) were highly expressed with 0.8 mM IPTG at 30℃and found in large amounts in crude cell extracts. the recombinant fusion protein were expressed under IPTG induction. Purified protein was used to immunize rabbit for the UL25 anti-serum preparation. Its agar diffusion reaction antibody titer was up to 1: 4. High specific IgG polyclonal antibody was obtained by purification using the caprylic acid and ammonium sulfate precipitation and High-Q anion-exchange chromatography.4. DPV UL25 gene product in DEF cells and cellular localization analysis Immunolocalization detection was observed using immunofluorescence technique, DPV UL25 gene expression product in the DEF in the cellular localization is a dynamic process. The results shown that specific fluorescence appeared relatively few in nucleus in 2 to 8 h and increased gradually in 24 to 48 h and eventually reached to the maximum, which aggregated in the spot region of the nucleus after the DEF were infected by DPV. However, there were only a small amount of specific fluorescences in the cytoplasm in 8 h and increased with the extension of infection time in 24 to 54 h. Then, the specific fluorescences finally reached to the maximum in the cytoplasm in 72 h. So, it can be infered from the distribution characteristics that DPV UL25 was synthetized in cytoplasm then transported to nucleus for the assembly of capsid precursor for the execution of its biological function.5. Transcription phase analysis of DPV UL25 gene The transcriptional analysis result of UL25 gene products in infected host cells detected by FQ-PCR result shows the DPV UL25 gene transcripts appeared as early as 1 h post infection, then the transcriptional products increased steadily and reached a peak at 12 hpi.Sequently, the transcriptional products lowed down to a certain degree at 12 hpi but maintained at a relative high level at 72 hpi, which owes the typical characterization of herpervirus late gene. Aggregate analysis the time course model of transcriptional and translational, it can be presume the DPV UL25 gene is a late gene, the expression of DPV UL25 may play an activation role in the assembly and maturation of DPV.