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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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The cloning strategy for constructing the UL55 recombinant plasmids. A. Schematic diagram of the UL55 ORF cloned into the pMD18-T cloning vector. B. Construction of the recombinant expression plasmid pET-32a(+)/UL55.
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Figure 1: The cloning strategy for constructing the UL55 recombinant plasmids. A. Schematic diagram of the UL55 ORF cloned into the pMD18-T cloning vector. B. Construction of the recombinant expression plasmid pET-32a(+)/UL55.

Mentions: Recombinant plasmids containing the encoding region of DEV UL55 were constructed for expression. Schematic diagrams of the cloning strategy of DEV UL55 were shown in Figure 1. The constructed recombinant plasmids pET32a(+)/UL55 was transformed into E. coli BL21 (DE3) for expression. After incubation at 37°C, the cultures were analyzed by SDS-PAGE. Results demonstrated that the E. coli BL21 (DE3) transformed with recombinant plasmid pET32a(+)/UL55 expressed a considerable amounts of a 40 KDa protein and it was mainly in the insoluble fraction(Figure 2, Lane 5). However, the corresponding band of pUL55 was absent in the inducing culture of pET32a(+) vector (Figure 2, Lane 1), the cultures of pET-32a(+)/UL55 before induction (Figure 2, Lane 2-3), and the supernatant of the culture of pET-32a(+)/UL55 after induction (Figure 2, Lane 4). Figure 3 indicated the optimal expression conditions of pUL55 in E. coli BL21 containing the working concentration of IPTG for inducing, the induction tempreture and the duration of IPTG. As a result, the maximum expression of pUL55 in prokaryotic system was induced by 0.2 mM IPTG (Figure 3A, Lane 5) at 37°C (Figure 3B, Lane 2) for 4.0 h (Figure 3C, Lane 1).


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)

The cloning strategy for constructing the UL55 recombinant plasmids. A. Schematic diagram of the UL55 ORF cloned into the pMD18-T cloning vector. B. Construction of the recombinant expression plasmid pET-32a(+)/UL55.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: The cloning strategy for constructing the UL55 recombinant plasmids. A. Schematic diagram of the UL55 ORF cloned into the pMD18-T cloning vector. B. Construction of the recombinant expression plasmid pET-32a(+)/UL55.
Mentions: Recombinant plasmids containing the encoding region of DEV UL55 were constructed for expression. Schematic diagrams of the cloning strategy of DEV UL55 were shown in Figure 1. The constructed recombinant plasmids pET32a(+)/UL55 was transformed into E. coli BL21 (DE3) for expression. After incubation at 37°C, the cultures were analyzed by SDS-PAGE. Results demonstrated that the E. coli BL21 (DE3) transformed with recombinant plasmid pET32a(+)/UL55 expressed a considerable amounts of a 40 KDa protein and it was mainly in the insoluble fraction(Figure 2, Lane 5). However, the corresponding band of pUL55 was absent in the inducing culture of pET32a(+) vector (Figure 2, Lane 1), the cultures of pET-32a(+)/UL55 before induction (Figure 2, Lane 2-3), and the supernatant of the culture of pET-32a(+)/UL55 after induction (Figure 2, Lane 4). Figure 3 indicated the optimal expression conditions of pUL55 in E. coli BL21 containing the working concentration of IPTG for inducing, the induction tempreture and the duration of IPTG. As a result, the maximum expression of pUL55 in prokaryotic system was induced by 0.2 mM IPTG (Figure 3A, Lane 5) at 37°C (Figure 3B, Lane 2) for 4.0 h (Figure 3C, Lane 1).

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