Search

Objective: The aim was to identify the codon usage bias between the newly identified duck plague virus (DPV) UL35 gene (GenBank accession No. EF643558) and the UL35-like genes of 27 other reference herpesviruses. Methods: A comparative analysis of the codon usage bias of the 28 herpesviruses was performed by using the CodonW 1.4 program and CUSP (create a codon usage table) program of EMBOSS (The European Molecular Biology Open Software Suite). Results: The results showed obvious differences of the synonymous codon usage bias in the 28 herpesviruses indicated by the Codon Adaptation Index, effective number of codons (ENc), and the value of G + C content at the 3rd codon position. The codon usage pattern of the DPV UL35 gene was phylogenetically conserved and similar to that of the UL35-like genes of the avian α-herpesvirus, with a strong bias towards the codons with A and T at the 3rd codon position. A cluster analysis of codon usage pattern of the DPV UL35 gene with other reference herpesviruses demonstrated that the codon usage bias of the UL35 genes of the 28 herpesviruses had a very close relation with their gene function. The ENc-plot revealed that the genetic heterogeneity in the DPV UL35 gene and the 27 reference herpesviruses were constrained by G + C content, while gene length exerted relatively weaker influences. In addition, comparisons of the codon preferences in the UL35 gene of DPV with those of Escherichia coli, yeast and humans revealed that there were 33 codons showing distinct usage differences between the DPV and yeast, and 38 between the DPV and humans, but only 31 between the DPV and E. coli. Therefore, the E. coli system may be more suitable for the expression of the DPV UL35 gene. Conclusion: Together, these results may improve our understanding of the evolution, pathogenesis and functional studies of DPV, as well as contribute significantly to the area of herpesvirus research and possibly studies with other viruses. Copyright © 2009 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Objective: Duck plague virus (DPV), the causative agent of duck plague (DP), is an alphaherpesvirus that causes an acute, febrile, contagious, and septic disease of waterfowl. UL35 protein (VP26) is a major capsid protein encoded by the UL35 gene, which is located in the unique long (UL) region of the DPV genome. To investigate the specific roles of VP26, the UL35 gene was amplified from the DPV DNA by polymerase chain reaction (PCR) and subcloned into pET-32a(+). Methods: The resultant prokaryotic expression vector, pET-32a(+)/UL35, includes an amino-terminal His6 as a fusion partner. Escherichia coli BL21 (DE3) competent cells were transformed with the construct and protein expression was subsequently induced by the addition of isopropyl-β-D-thiogalactopyranoside to the culture medium. Protein lysates were submitted to SDS-PAGE to evaluate recombinant protein expression. Results: The band that corresponded to the predicted protein size (33 kDa) was observed on the SDS-PAGE gel. The recombinant His6-tagged VP26 fusion protein was expressed at a high level in an insoluble form (inclusion bodies) and constituted about 24% of the total cellular protein. Then, the fusion protein was purified to near homogeneity using single-step immobilized metal affinity chromatography on a nickel-nitrilotriacetic acid affinity resin, yielding about 620 mg per liter culture. After purification, New Zealand white rabbits were immunized with purified His6-tagged VP26 in order to raise polyclonal antibody against this recombinant protein. Using the resultant sera, Western blot analysis showed that the recombinant protein was recognized by the polyclonal antibody. Conclusion: Thus, the polyclonal antibody prepared here may serve as a valuable tool to study the functional involvement of VP26 in the DPV life cycle. Copyright © 2009 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]