Abstract
BackgroundThe UL54 protein of Duck Enteritis Virus (DEV) is a homolog of herpes simplex virus-1 (HSV-1) immediate-early infectious cell protein 27 (ICP27), a multifunctional protein essential for viral infection. Nonetheless, there is little information on the UL54 protein of DEV.MethodsThe UL54 gene was cloned into the pPAL7 vector, and the recombinant protein, expressed in the E. coli Rosetta, was used to produce a specific antibody. Using this antibody, Western blotting and indirect immunofluorescence analysis (IFA) were used to analyze the expression level and intracellular localization, respectively, of UL54 in DEV-infected cells at different times. Real-time quantitative reverse transcription PCR (RT-PCR) and the pharmacological inhibition test were utilized to ascertain the kinetic class of the UL54 gene.ResultsUL54 was expressed as a fusion protein of approximately 66.0 kDa using the prokaryotic expression system, and this protein was used to generate the specific anti-UL54 antibody. The UL54 protein was initially diffusely distributed throughout the cytoplasmic region; then, after 2 h, it gradually distributed into the nucleus, peaking at 24 h, and complete localization to the nucleus was observed thereafter. The UL54 transcript was detected as early as 0.5 h, and peak expression was observed at 24 h. The UL54 gene was insensitive to the DNA polymerase inhibitor Ganciclovir (GCV) and the protein synthesis inhibitor Cycloheximide (CHX), both of which confirmed that UL54 was an immediate early gene.ConclusionsThe DEV UL54 gene was expressed in a prokaryotic expression system and characterized for expression level, intracellular localization and gene kinetic class. We propose that these results will provide the foundation for further functional analyses of this gene.
Highlights
The UL54 protein of Duck Enteritis Virus (DEV) is a homolog of herpes simplex virus-1 (HSV-1) immediate-early infectious cell protein 27 (ICP27), a multifunctional protein essential for viral infection
The DEV UL54 protein was expressed in an E. coli expression system The UL54 gene was cloned into vector pPAL7 and expressed under varying conditions, including different E. coli host cells, inducer concentrations, induction temperatures and induction durations (Fig. 1)
The UL54 protein was expressed in E. coli Rosetta under optimized conditions and purified through gel and electric elution (Fig. 2a)
Summary
Viruses and cells DEV CHV strain was separated and preserved in the laboratory. Monolayer cultures of duck embryo fibroblast (DEF) cells were grown in Modified Eagle’s medium (MEM) supplemented with 10 % newborn calf serum (NBS) at 37 °C in a 5 % CO2 humidified incubator. Western blotting The UL54 protein expression in E. coli Rosetta and DEF cells were detected using western blotting with rabbit anti-DEV and anti-DEV UL54 protein antibodies, respectively, as the primary antibodies [33]. A standard protocol was performed after the proteins were separated through electrophoresis on 12 % SDS-PAGE gels and transferred to polyvinylidene fluoride (PVDF) membranes using a semi-dry transfer cell apparatus. The membranes were blocked with PBS buffer containing 1 % BSA for 1 h and subsequently incubated with the diluted primary antibody for an additional 1 h. The membrane was washed for 30 min with 1× PBS containing 0.05 % Tween-20 and subsequently incubated with goat anti-rabbit HRP-labeled IgG secondary antibodies for 30 min. Total RNA was isolated from DEV-infected DEF cells incubated with GCV or CHX at 24 h post-infection and subsequently reverse transcribed into cDNA.
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