Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Wenjiang, Chengdu city, Sichuan, 611130, PR China.
Virol J. 2011 May 24;8:256. doi: 10.1186/1743-422X-8-256.
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.
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.
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.
新鉴定的鸭肠炎病毒 UL55 基因产物的特征尚未报道。对 UL55 蛋白的了解可以提供其功能的有用见解。
新鉴定的鸭肠炎病毒 UL55 基因约 561bp，经扩增并消化构建重组质粒 pET32a(+)/UL55，在大肠杆菌中表达。SDS-PAGE 分析显示，在 37°C 下用 0.2mM IPTG 诱导 4 小时后，重组蛋白 UL55(pUL55)在大肠杆菌 BL21 宿主细胞中大量表达，并聚集为包涵体。变性后约 40kDa 的蛋白 pUL55 经五次洗涤纯化，并用其免疫兔制备多克隆抗体。用琼脂免疫扩散和中和试验检测和鉴定了制备的针对 pUL55 的多克隆抗体。Western blot 分析和细胞内分析结果表明，pUL55 在复制晚期表达最丰富，主要分布在鸭肠炎病毒感染细胞的细胞质中。
本研究成功地在原核表达系统中表达了鸭肠炎病毒 UL55 蛋白。此外，我们已经制备了针对重组蛋白 UL55 的多克隆抗体，并首次对鸭肠炎病毒 UL55 蛋白的一些特性进行了表征。该研究将有助于进一步分析该基因的功能。
