Avian Diseases Research Center, College of Veterinary Medicine of Sichuan Agricultural University, Yaan, Sichuan, China.
Virol J. 2010 Jun 8;7:120. doi: 10.1186/1743-422X-7-120.
The gE protein of duck plague virus is the important membrane glycoprotein, its protein characterization has not been reported. In this study, we expressed and presented the characterization of the DPV gE product.
According to the sequence of the gE gene, a pair of primers were designed, and the DNA product with 1490bp in size was amplified by using the polymerase chain reaction (PCR). The PCR product was cloned into pMD18-T vector, and subcloned into pET32a(+), generating the recombinant plasmid pET32a/DPV-gE. SDS-PAGE analysis showed that the fusion pET32a/DPV-gE protein was highly expressed after induction by 0.2 mM IPTG at 30 degrees C for 4.5 h in Rosseta host cells. Over expressed 6xHis-gE fusion protein was purified by nickel affinity chromatography, and used to immunize the rabbits for the preparation of polyclonal antibody. The result of the intracellular localization revealed that the gE protein was appeared to be in the cytoplasm region. The real time PCR, RT-PCR analysis and Western blotting revealed that the gE gene was produced most abundantly during the late phase of replication in DPV-infected cells.
In this work, the DPV gE protein was successfully expressed in a prokaryotic expression system, and we presented the basic properties of the DPV gE product for the first time. These properties of the gE protein provided a prerequisite for further functional analysis of this gene.
鸭瘟病毒的 gE 蛋白是重要的膜糖蛋白，其蛋白特性尚未报道。本研究中，我们表达并呈现了 DPV gE 产物的特性。
根据 gE 基因序列，设计了一对引物，通过聚合酶链反应（PCR）扩增出大小为 1490bp 的 DNA 产物。将 PCR 产物克隆到 pMD18-T 载体中，并亚克隆到 pET32a(+)中，生成重组质粒 pET32a/DPV-gE。SDS-PAGE 分析表明，在罗斯塔宿主细胞中，用 0.2mM IPTG 在 30°C 诱导 4.5 小时后，融合 pET32a/DPV-gE 蛋白高度表达。通过镍亲和层析纯化过表达的 6xHis-gE 融合蛋白，并用于免疫兔子制备多克隆抗体。细胞内定位结果表明，gE 蛋白似乎位于细胞质区域。实时 PCR、RT-PCR 分析和 Western blotting 表明，在 DPV 感染细胞中，gE 基因在复制后期表达最丰富。
在这项工作中，DPV gE 蛋白在原核表达系统中成功表达，我们首次呈现了 DPV gE 产物的基本特性。这些 gE 蛋白的特性为进一步分析该基因的功能提供了前提条件。
