Generation of Pseudorabies-resistant Animals by Germ-line Transformation

为了检测伪狂犬病毒（PRV）几种基因启动子在体内的活性，我们在病毒启动子的控制下产生了表达转基因基因的转基因小鼠。转基因表达的组织特异性分析表明，即早基因启动子是神经元组织特异性的，早期蛋白0基因启动子是泛特异性的，而潜伏期相关转录基因启动子是神经元特异性的。为了在体内评估PRV IE180和EP0显性阴性突变体的抗病毒潜力，我们制备了几种表达IE180或EP0显性阴性突变体的转基因小鼠系。目前正在对这些转基因小鼠进行实验感染。疱疹病毒进入介质（HVEM）是肿瘤坏死因子受体家族的一员，被单纯疱疹病毒（HSV）的病毒粒子糖蛋白D （gD）用作细胞受体。为了评估HVEM在体内的抗病毒潜力，我们培育了3株表达HVEM可溶性形式（HVEMlg）的转基因小鼠系，该形式由小鼠HVEM的细胞外结构域和人lgG1的Fc部分组成。所有转基因小鼠系对HSV-1感染均表现出明显的抗性。为了使这一发现适用于伪狂犬病耐药动物的产生，我们使用了猪疱疹病毒进入介质C （HveC）。用表达猪HveC胞外结构域和人lgG1 Fc部分的融合蛋白的嵌合基因转染Vero细胞。表达融合蛋白的转化细胞系对PRV感染表现出明显的抗性。表达融合蛋白的转基因小鼠的产生目前正在进行中。这些发现表明，该融合蛋白在提高家畜对伪狂犬的抗病能力方面具有潜在价值。

To examine the activity of several gene promoters of pseudorabies virus (PRV) in vivo, we have generated transgenic mice expressing transgenes under the control of the viral promoters. Analyses of the tissue specificity of transgene expression demonstrated that the immediate-early gene promoter is neuronal tissue-specific, the early protein 0 gene promoter is a pan-specific and the latency associated transcript gene promoter is a neuron-specific, respectively.In order to assess the antiviral potential of PRV IE180 and a dominant-negative mutant of EP0 in vivo, several transgenic mouse lines expressing IE180 or the dominant-negative mutant of EP0 were generated. Experimental infection to these transgenic mice is now in progress.Herpesvirus entry mediator (HVEM) is a member of the tumor necrosis factor receptor family used as a cellular receptor by virion glycoprotein D (gD) of herpes simplex virus (HSV). In order to assess the antiviral potential of HVEM in vivo, three transgenic mouse lines expressing a soluble form of HVEM (HVEMlg) consisting of an extracellular domain of murine HVEM and the Fc portion of human lgG1 were generated. All of the transgenic mouse lines showed marked resistance to HSV-1 infection. To adapt this finding for generation of pseudorabies-resistant animal, we used porcine herpesvirus entry mediator C (HveC). Vero cells were transformed with the chimeric gene expressing a fusion protein consisting of an extracellular domain of porcine HveC and the Fc portion of human lgG1. The transformed cell lines expressing the fusion protein showed marked resistance to PRV infection. Generation of transgenic mice expressing the fusion protein is now in progress. These findings suggest the potential value of the fusion protein in agricultural livestock for enhanced disease resistance to pseudorabies.