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Characterization of the duck enteritis virus UL55 protein.

Wu Y, Cheng A, Wang M, Zhang S, Zhu D, Jia R, Luo Q, Chen Z, Chen X - Virol. J. (2011)

Bottom Line: The denatured protein about 40 KDa named pUL55 was purified by washing five times, and used to immune rabbits for preparation of polyclonal antibody.The prepared polyclonal antibody against pUL55 was detected and determined by Agar immundiffusion and Neutralization test.The research will be useful for further functional analysis of this gene.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Wenjiang, Chengdu city, Sichuan, 611130, PR China.

ABSTRACT

Background: Characteration of the newly identified duck enteritis virus UL55 gene product has not been reported yet. Knowledge of the protein UL55 can provide useful insights about its function.

Results: The newly identified duck enteritis virus UL55 gene was about 561 bp, it was amplified and digested for construction of a recombinant plasmid pET32a(+)/UL55 for expression in Escherichia coli. SDS-PAGE analysis revealed the recombinant protein UL55(pUL55) was overexpressed in Escherichia coli BL21 host cells after induction by 0.2 mM IPTG at 37°C for 4 h and aggregated as inclusion bodies. The denatured protein about 40 KDa named pUL55 was purified by washing five times, and used to immune rabbits for preparation of polyclonal antibody. The prepared polyclonal antibody against pUL55 was detected and determined by Agar immundiffusion and Neutralization test. The results of Wstern blotting assay and intracellular analysis revealed that pUL55 was expressed most abundantly during the late phase of replication and mainly distributed in cytoplasm in duck enteritis virus infected cells.

Conclusions: In this study, the duck enteritis virus UL55 protein was successfully expressed in prokaryotic expression system. Besides, we have prepared the polyclonal antibody against recombinant prtein UL55, and characterized some properties of the duck enteritis virus UL55 protein for the first time. The research will be useful for further functional analysis of this gene.

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Intracellular localization of DEV pUL55 in DEV-infected cells at 5.5 h, 49 h, 54 h post-infection. J-L. DEV-infected cells were fixed at 45.5 h, 49 h, 54 h post-infection. Samples were incubated with anti-pUL55 serum and subsequently stained with fluorescein isothiocyanate(FITC)-conjugated secondary antibody. Nuclei were counterstained with DAPI(blue). The merged fluorescence microscopy image of DEF are shown in panels J-L with high magnification (600×).
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Figure 13: Intracellular localization of DEV pUL55 in DEV-infected cells at 5.5 h, 49 h, 54 h post-infection. J-L. DEV-infected cells were fixed at 45.5 h, 49 h, 54 h post-infection. Samples were incubated with anti-pUL55 serum and subsequently stained with fluorescein isothiocyanate(FITC)-conjugated secondary antibody. Nuclei were counterstained with DAPI(blue). The merged fluorescence microscopy image of DEF are shown in panels J-L with high magnification (600×).

Mentions: The intracellular distribution of pUL55 in DEV infected cells was examined by indirect immunofluorescence staining with purified anti-pUL55 serum. At various times after infection, DEF cells were collected and fixed in cold paraformaldehyde. Optimization results revealed the coverslips were expected to be fixed at 4°C overnight with 4% cold paraformaldehyde, and then treated with 4% BSA to block the nonspecific staining, the permeabilization time was with 0.2% (v/v) TrionX-100 in PBS for an additional 30 min at 4°C and the anti-pUL55 IgG was supposed to diluted 1:64 to incubate at 4°C overnight in the coverslips (datas not shown). As shown in Figure 10C, the pUL55 was distributed in bright fluorescent granules in the cytoplasm of infected cells at 5.5 h p.i. However, these fluorescence pellets were absent from mock-infected cells (Figure 10A), and no significant fluorescence was observed with the preimmune serum(Figure 10B). After that, the detectable fluoresecence structures kept increasing, the strongest fluorescence was observed at 22.5 h p.i (Figure 11F). From Figure 10C to Figure 12H, we easily found the bringht fluorescence granules were widely distributed in the cytoplasm and gradually near the periphery of the nucleus even traces of them within nuclear. Starting from 40 h p.i, the fluorescence granules expressed diffusely throughuout the cytoplasm then reclustered to bright speckled structures which distributed especially in the juxtanuclear region (from Figure 13J to Figure 14M). These fluorescence gradually diminished as time going on. Meanwhile, the DEF cells turned into the round shape and began to shed off at 54 h p.i. At 74 h post infection (Figure 14O), the pUL55-specific fluorescence almost vanish following the cytoplasm disintegration in infected cells.


Characterization of the duck enteritis virus UL55 protein.

Wu Y, Cheng A, Wang M, Zhang S, Zhu D, Jia R, Luo Q, Chen Z, Chen X - Virol. J. (2011)

Intracellular localization of DEV pUL55 in DEV-infected cells at 5.5 h, 49 h, 54 h post-infection. J-L. DEV-infected cells were fixed at 45.5 h, 49 h, 54 h post-infection. Samples were incubated with anti-pUL55 serum and subsequently stained with fluorescein isothiocyanate(FITC)-conjugated secondary antibody. Nuclei were counterstained with DAPI(blue). The merged fluorescence microscopy image of DEF are shown in panels J-L with high magnification (600×).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3117846&req=5

Figure 13: Intracellular localization of DEV pUL55 in DEV-infected cells at 5.5 h, 49 h, 54 h post-infection. J-L. DEV-infected cells were fixed at 45.5 h, 49 h, 54 h post-infection. Samples were incubated with anti-pUL55 serum and subsequently stained with fluorescein isothiocyanate(FITC)-conjugated secondary antibody. Nuclei were counterstained with DAPI(blue). The merged fluorescence microscopy image of DEF are shown in panels J-L with high magnification (600×).
Mentions: The intracellular distribution of pUL55 in DEV infected cells was examined by indirect immunofluorescence staining with purified anti-pUL55 serum. At various times after infection, DEF cells were collected and fixed in cold paraformaldehyde. Optimization results revealed the coverslips were expected to be fixed at 4°C overnight with 4% cold paraformaldehyde, and then treated with 4% BSA to block the nonspecific staining, the permeabilization time was with 0.2% (v/v) TrionX-100 in PBS for an additional 30 min at 4°C and the anti-pUL55 IgG was supposed to diluted 1:64 to incubate at 4°C overnight in the coverslips (datas not shown). As shown in Figure 10C, the pUL55 was distributed in bright fluorescent granules in the cytoplasm of infected cells at 5.5 h p.i. However, these fluorescence pellets were absent from mock-infected cells (Figure 10A), and no significant fluorescence was observed with the preimmune serum(Figure 10B). After that, the detectable fluoresecence structures kept increasing, the strongest fluorescence was observed at 22.5 h p.i (Figure 11F). From Figure 10C to Figure 12H, we easily found the bringht fluorescence granules were widely distributed in the cytoplasm and gradually near the periphery of the nucleus even traces of them within nuclear. Starting from 40 h p.i, the fluorescence granules expressed diffusely throughuout the cytoplasm then reclustered to bright speckled structures which distributed especially in the juxtanuclear region (from Figure 13J to Figure 14M). These fluorescence gradually diminished as time going on. Meanwhile, the DEF cells turned into the round shape and began to shed off at 54 h p.i. At 74 h post infection (Figure 14O), the pUL55-specific fluorescence almost vanish following the cytoplasm disintegration in infected cells.

Bottom Line: The denatured protein about 40 KDa named pUL55 was purified by washing five times, and used to immune rabbits for preparation of polyclonal antibody.The prepared polyclonal antibody against pUL55 was detected and determined by Agar immundiffusion and Neutralization test.The research will be useful for further functional analysis of this gene.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Wenjiang, Chengdu city, Sichuan, 611130, PR China.

ABSTRACT

Background: Characteration of the newly identified duck enteritis virus UL55 gene product has not been reported yet. Knowledge of the protein UL55 can provide useful insights about its function.

Results: The newly identified duck enteritis virus UL55 gene was about 561 bp, it was amplified and digested for construction of a recombinant plasmid pET32a(+)/UL55 for expression in Escherichia coli. SDS-PAGE analysis revealed the recombinant protein UL55(pUL55) was overexpressed in Escherichia coli BL21 host cells after induction by 0.2 mM IPTG at 37°C for 4 h and aggregated as inclusion bodies. The denatured protein about 40 KDa named pUL55 was purified by washing five times, and used to immune rabbits for preparation of polyclonal antibody. The prepared polyclonal antibody against pUL55 was detected and determined by Agar immundiffusion and Neutralization test. The results of Wstern blotting assay and intracellular analysis revealed that pUL55 was expressed most abundantly during the late phase of replication and mainly distributed in cytoplasm in duck enteritis virus infected cells.

Conclusions: In this study, the duck enteritis virus UL55 protein was successfully expressed in prokaryotic expression system. Besides, we have prepared the polyclonal antibody against recombinant prtein UL55, and characterized some properties of the duck enteritis virus UL55 protein for the first time. The research will be useful for further functional analysis of this gene.

Show MeSH
Related in: MedlinePlus