A new approach for the control of Aujeszky's disease by using germ-line transformation

利用病毒蛋白的显性阴性突变体进行细胞内免疫被认为是人类抗病毒治疗和动物种系转化以获得病毒感染抗性的新方法。为了获得有效的Aujeszky's disease virus （ADV）即刻早期（IE）基因表达抑制因子，构建了编码ADV IE蛋白（IE180）和早期蛋白0 （EPO）显性阴性突变体的突变基因，以及编码由IE180 dna结合域和缺乏转录激活域的尾状单纯疱疹病毒1 VP16组成的融合蛋白的嵌合基因。在瞬时表达实验中，这些基因产物抑制了ADV IE启动子的转录。这些基因稳定转化的HeLa细胞株显示出对ADV感染的抗性。其中，用嵌合转基因转化的细胞系的抗性显著。为了评估融合蛋白在体内的抗病毒潜力，我们使用多肽链延伸因子1α或小鼠Mx1启动子控制的嵌合基因来产生转基因小鼠。在C57BL/6受精卵中注入约1000份编码嵌合基因的DNA片段。在41只出生的动物中，有5只动物通过尾部DNA的Southern blot分析确定了转基因。四位创始人未能将转基因基因传给后代。剩下的一株将转基因遗传给了F1后代。在转基因小鼠中，融合蛋白在Mx1启动子的控制下表达，Mx1启动子可被双链RNA、干扰素或病毒感染诱导。为了评估建立的转基因小鼠对ADV的抗性，F1小鼠的育种正在进行中。

Intracellular immunization using dominant-negative mutants of viral proteins is prorposed as a new approach to antiviral therapy in humans and to germ-line transformation in animals to confer resistance to viral infections. To obtain effective repressors of Aujeszky's disease virus (ADV) immediate-early (IE) gene expression, mutant genes encoding dominant-negative mutants of ADV IE protein (IE180) and early protein 0 (EPO), and a chimeric gene encoding a fusion protein consisting of the DNA-binding domain of IE180 and a tail-trucated herpes simplex virus 1 VP16 lacking the transcription activation domain were constructed. These gene products inhibited transcription from the ADV IE promoter in transient expression assays. HeLa cell lines stably transformed with the genes showed resistance to ADV infection. Among them, resistance of a cell line transformed with the chimeric transgene was remarkable.In order to assess the antiviral potential of the fusion protein in vivo, the chimeric gene under the control of polypepetide chain elongation factor 1alpha or the mouse Mx1 promoter was used for generation of transgenic mice. C57BL/6 zygotes were microinjected with about 1000 copies of the DNA fragment encoding the chimeric gene. Of the resulting 41 births, five animals had the transgene as determined by Southern blot analysis of tail DNA.Four of the founders failed to transmit the transgene to their progeny. A remaining line transmitted the transgene to F1 progeny. In the transgenic mice, the fusion protein is expressed under the control of the Mx1 promoter which is inducible by double stranded RNA,interferon, or virus infection. To assess the resistance of the established transgenic mice to ADV challenge, breeding of the F1 mice is now in progress.