His6-tagged UL35 protein of duck plague virus: expression, purification, and production of polyclonal antibody.

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-beta-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.

Characterization of synonymous codon usage bias in the duck plague virus UL35 gene.

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 alpha-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.

Identification and characterization of the duck enteritis virus UL51 gene.

Compared to the UL51 gene of other alphaherpesviruses, the duck enteritis virus (DEV) UL51 gene contains ten conserved motifs and has a close evolutionary relationship with members of the genus Mardivirus. The DEV UL51 gene product was identified using a rabbit polyclonal antiserum raised against a 6-His-UL51 fusion protein expressed in Escherichia coli as a 34-kDa protein. Western blotting and RT-(real time) PCR analysis of DEV-infected cells showed that the protein was produced at the late stage of infection and that its production was highly dependent on viral DNA synthesis, suggesting that the gene should be classified as gamma2 class. Analysis of extracellular virions revealed that the protein was a component of extracellular mature DEV virions. Indirect immunofluorescence studies localized most of the protein to the juxtanuclear region. These results will provide a basis for further functional analysis of the gene.

Identification and characterization of duck enteritis virus dUTPase gene.

Deoxyuridine triphosphatase (dUTPase) is a ubiquitous and important enzyme that hydrolyzes dUTP to dUMP. Many viruses encode virus-specific dUTPase, which plays an essential role in maintaining the integrity of the viral DNA both by reducing the dUTP levels and by providing the substrate for the thymidylate synthase. A 1344-bp gene of duck enteritis virus (DEV) homologous to herpesviral dUTPase was first reported in this paper. The gene encodes a protein of 477 amino acids, with a predicted molecular mass of 49.7 kDa. Multiple sequence alignment suggested that DEV dUTPase was quite similar to other identified herpesviral dUTPase and functioned as a homotrimer. The five conserved motifs of DEV dUTPase with 3-1-2-4-5 arrangement have been recognized, and the phylogenetic analysis showed that DEV dUTPase was genetically close to the avian herpesvirus. Furthermore, RNA dot blot, western blot, and immunofluorescence analysis indicated that the enzyme was expressed at early and late stages after infection. Immunofluorescence also confirmed that DEV dUTPase localized in the cytoplasm of DEV-infected duck embryo fibroblasts as early as 4 hr postinfection (hpi). Later, the enzyme transferred from cytoplasm to nucleus at 8 hpi, and then reached its expression peak at 12 hpi, both in the cytoplasm and nucleus. The results suggested that the DEV dUTPase gene might be an early viral gene in DEV vitro infection and contribute to ensuring the fidelity of genome replication.

[Cloning, prokaryotic expression and subcellular localization in the infected host cells of the duck plague virus DPV UL35 gene].

Based on the duck plague virus (DPV) UL35 gene sequence that our laboratory obtained (GenBank accession number EF643558), a pair of primers was designed using Oligo6.0 and primer5.0, then the UL35 gene was amplified from DPV CHv strain genomic DNA and cloned into the pMD18-T to construct a clone plasmid pMD18-T-UL35. After identification of the pMD18-T-UL35 by PCR amplification and restriction digestion, the fragment of the UL35 gene was subcloned into the prokaryotic expression vector pET-32a(+). The resultant recombinant plasmid pET-32a(+)-UL35 was then transformed into E. coli BL21 (DE3) strain and optimally-expressed under the induction of 1.0 mmol/L IPTG at 34 degrees C for 5 hours. SDS-PAGE analysis showed the recombinant protein (VP26) had a molecular weight of about 33KDa and accounted for 32.3% of total bacterial protein by gel scanning. The protein was then purified by Ni(2+)-affinity chromatography and used to immunize rabbit for producing the VP26 anti-serum and its antibody titer was up to 1:32 detected by agar diffusion reaction. After the IgG of the polyclonal antibodies was purified by High-Q anion-exchange chromatography, Western blot analysis indicated that the IgG had specific reaction with the VP26. Moreover, the subcellular localization detection was observed using immunofluorescence technique. The results showed that the specific fluorescences appeared relatively few in nucleus in 2 to 8 hours and increased gradually in 12 to 36 hours and eventually reached to the maximum, which aggregated in the spot region of the nucleus after the duck embryo fibroblast (DEF) were infected by DPV. However, there were only a small amount of specific fluorescences in the cytoplasm in 12 hours and increased with the extension of infection time in 24 to 48 hours. The specific fluorescences finally reached to the maximum in the cytoplasm in 72 hours. The results provided significant data for furthering the study on the function of DPV UL35 gene.