Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Wenjiang, Chengdu city, Sichuan, 611130, PR China.
Virol J. 2011 May 19;8:241. doi: 10.1186/1743-422X-8-241.
At present, alphaherpesviruses gI gene and its encoding protein have been extensively studied. It is likely that gI protein and its homolog play similar roles in virions direct cell-to-cell spread of alphaherpesviruses. But, little is known about the characteristics of DEV gI gene. In this study, we expressed and presented the basic properties of the DEV gI protein.
The special 1221-bp fragment containing complete open reading frame(ORF) of duck enteritis virus(DEV) gI gene was extracted from plasmid pMD18-T-gI, and then cloned into prokaryotic expression vector pET-32a(+), resulting in pET-32a(+)-gI. After being confirmed by PCR, restriction endonuclease digestion and sequencing, pET-32a(+)-gI was transformed into E.coli BL21(DE3) competent cells for overexpression. DEV gI gene was successfully expressed by the addition of isopropyl-β-D-thiogalactopyranoside(IPTG). SDS-PAGE showed that the recombinant protein His6-tagged gI molecular weight was about 61 kDa. Subsequently, the expressed product was applied to generate specific antibody against gI protein. The specificity of the rabbit immuneserum was confirmed by its ability to react with the recombinant protein His6-tagged gI. In addition, real time-PCR was used to determine the the levels of the mRNA transcripts of gI gene, the results showed that the DEV gI gene was transcribed most abundantly during the late phase of infection. Furthermore, indirect immunofluorescence(IIF) was established to study the gI protein expression and localization in DEV-infected duck embryo fibroblasts (DEFs), the results confirmed that the protein was expressed and located in the cytoplasm of the infected cells, intensively.
The recombinant prokaryotic expression vector of DEV gI gene was constructed successfully. The gI protein was successfully expressed by E.coli BL21(DE3) and maintained its antigenicity very well. The basic information of the transcription and intracellular localization of gI gene were presented, that would be helpful to assess the possible role of DEV gI gene. The research will provide useful clues for further functional analysis of DEV gI gene.
目前，α疱疹病毒 gI 基因及其编码蛋白已得到广泛研究。gI 蛋白及其同源物很可能在α疱疹病毒的病毒粒子与细胞之间的直接传播中发挥相似的作用。但是，关于 DEV gI 基因的特征知之甚少。在本研究中，我们表达并展示了 DEV gI 蛋白的基本特性。
从质粒 pMD18-T-gI 中提取了包含鸭肠炎病毒(DEV)gI 基因完整开放阅读框(ORF)的特殊 1221bp 片段，然后将其克隆到原核表达载体 pET-32a(+)中，得到 pET-32a(+)-gI。经 PCR、限制性内切酶消化和测序验证后，将 pET-32a(+)-gI 转化入 E.coli BL21(DE3)感受态细胞中进行过表达。加入异丙基-β-D-硫代半乳糖苷(IPTG)后，DEV gI 基因成功表达。SDS-PAGE 显示，重组蛋白 His6 标记的 gI 分子量约为 61kDa。随后，该表达产物被用于产生针对 gI 蛋白的特异性抗体。兔免疫血清与重组蛋白 His6 标记的 gI 的反应能力证实了其特异性。此外，实时 PCR 用于确定 gI 基因的 mRNA 转录水平，结果表明 DEV gI 基因在感染的晚期转录最丰富。此外，建立间接免疫荧光(IIF)来研究 DEV 感染鸭胚成纤维细胞(DEFs)中的 gI 蛋白表达和定位，结果证实该蛋白在感染细胞的细胞质中表达并强烈定位。
成功构建了 DEV gI 基因的重组原核表达载体。大肠杆菌 BL21(DE3)成功表达了 gI 蛋白，并很好地保持了其抗原性。提出了 gI 基因转录和细胞内定位的基本信息，这有助于评估 DEV gI 基因的可能作用。该研究将为进一步分析 DEV gI 基因的功能提供有用的线索。
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