Molecular characterization of duck enteritis virus UL41 protein

Figures

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  Fig. 1. Purification of the fusion protein and preparation of the polyclonal antibody. All bacteria samples were collected by centrifugation at 4 °C and disrupted by ultrasonication, and then analyzed by SDS-PAGE with 12% resolving gels and 5% stacking gels. a Lane 1, pET-32a(+) vector; Lane 2, recombinant bacterial sediment; Lane 3, recombinant bacterial supernatant; M. Bio-Rad low-molecular-weight protein marker. Protein expression was induced at 37 °C for 6 h with 0.4 mM IPTG until OD600 reached 0.4–0.6. Significantly higher UL41-His recombinant protein levels were observed in the recombinant bacterial sediment. b IPTG concentration optimization. Lanes 1–7, IPTG concentrations of 1.2, 1.0, 0.8, 0.6, 0.4, 0.2, and 0.1 mM, respectively. M. Bio-Rad low-molecular-weight protein marker. Significantly higher UL41-His recombinant protein levels were observed at 0.2 mM. c Induction temperature optimization. Lane 1, induction at 25 °C; Lane 2, induction at 30 °C; Lane 3, induction at 37 °C; M. Bio-Rad low-molecular-weight protein marker. Significantly higher UL41-His recombinant protein levels were observed at 30 °C. d Induction time optimization. Lanes 1–7, induction for 12.5, 10, 8, 6, 4, 2,…

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  Fig. 2. The transcription and expression kinetics of the DEV UL41 gene. a Melting curves of DEV UL41 (81 °C) and β-actin (89 °C). b Standard curves of DEV UL41 (Y = − 3.271X + l.048) and β-actin (Y = − 3.275X + 0.978), and UL41 gene (102.2%) and β-actin (102.0%) amplification efficiencies were approximately identical, with correlation coefficients of 0.999. c UL41 mRNA levels at 0, 2, 6, 12, 24, 36, 42, 45, 48, and 54 hpi were assessed by RT-qPCR and normalized toβ-actin. The UL41 transcript was detected at 6 hpi, increased gradually (6–30 hpi), reached its peak at 36 hpi, and then decreased steadily thereafter (42–54 hpi). d Representative western blotting results for UL41 at 7, 24, 36, 48, 60, and 72 hpi (up) (approximately 56 kDa) and quantification in the six groups (down). The negative group comprised mock-treated DEF cells detected with mouse anti-UL41 antibody serum (1:800) and goat anti-mouse IgG HRP-conjugated antibody (1:5000). The band density increased gradually (7-36hpi), peak expression occurred at 48 hpi, and expression decreased steadily thereafter (60–72 hpi). β-actin antibody was used to assess β-actin as the loading control

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  Fig. 3. Pharmaceutical identification virion gene type. a M, DL2000 Marker; ACV group: DEV-infected cells treated with 300 μg/ml acyclovir (ACV); CHX group: DEV-infected cells treated with 50 μg/mL cycloheximide (CHX). (+) group: DEV-infected cells without drugs; (−) group: untreated DEF cells. The DEV IE gene UL54 (127 bp), E gene UL13 (131 bp), and L gene Us2 (111 bp) are shown, respectively. b M, DL2000 Marker; housekeeping gene β-actin (177 bp) and DEV UL41 (1526 bp) are shown, respectively

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  Fig. 4. pUL41 intracellular localization in DEV-infected cells at different times. a Representative confocal immunofluorescence microscopic images of the UL41 localization (green in all images) at 7, 12, 24, 36, 48, 60, and 72 hpi. Nuclei are indicated in blue (DAPI). The control group comprised untreated DEF cells (magnification: 200×; scale bar: 50 μm) detected with mouse anti-UL41 antibody serum (1:800) and goat anti-mouse IgG (H + L) cross-adsorbed secondary antibody, Alexa Fluor 488 (1:1000). Significantly higher UL41 levels in the cytoplasm were evident in the DEF cells infected with wild-type DEV

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  Fig. 5. DEV-UL47 protein may translocate pUL41 into the nucleus in the absence of other viral proteins. All transfected samples were collected after 24 h post transfection. a DEF cells were transfected with pEGFP-C2-UL41 and pEGFP-C2, respectively. The EGFP-UL41 fusion protein was localized to the cytoplasm (green), further detected with mouse anti-UL41 protein polyclonal antibody (1:800) and goat anti-mouse IgG (H + L) cross-adsorbed secondary antibody, Alexa Fluor 568 (1:1000) (red). The pEGFP-C2 plasmid control was distributed throughout the cells (green). b DEF cells were co-transfected with pEGFP-C2-UL41 and pcDNA3.1-UL47. Much of the EGFP-UL41 fusion protein was localized to the nucleus (green), and pUL47 was mainly localized to the nucleus (red), detected with the rabbit anti-UL47 protein polyclonal antibody (1:1000) and goat anti-rabbit IgG (H + L) cross-adsorbed secondary antibody, Alexa Fluor 568 (1:1000). DEF cells were co-transfected with pEGFP-C2-UL41 and pcDNA3.1(+) as the control, and the EGFP-UL41 fusion protein was only distributed in the cytoplasm (green). c DEF cells were transfected with pcDNA3.1-UL47Δ40–50 and pcDNA3.1-UL47Δ768–777, respectively. A portion of the…