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

利用病毒蛋白显性失活突变体的细胞内免疫被认为是人类抗病毒治疗和动物生殖系转化以赋予对病毒感染的抗性的新方法。为了获得有效的抑制Aujeszky病病毒（ADV）即早期（IE）基因表达的基因，构建了ADV IE蛋白（IE 180）和早期蛋白0（EPO）显性负突变体的突变基因，以及IE 180的DNA结合域与缺失转录激活域的单纯疱疹病毒1型VP 16的融合基因。这些基因产物在瞬时表达测定中抑制ADV IE启动子的转录。用该基因稳定转化的HeLa细胞系显示出对ADV感染的抗性。为了评估融合蛋白在体内的抗病毒潜力，将多肽链延伸因子1 α或小鼠Mx 1启动子控制下的嵌合基因用于产生转基因小鼠。用约1000个拷贝的编码嵌合基因的DNA片段显微注射C57 BL/6合子。通过对尾部DNA的Southern印迹分析，在最终的41只后代中，有5只带有转基因，其中4只没有将转基因传递给后代。剩余的系将转基因传递到F1子代。在转基因小鼠中，融合蛋白在Mx 1启动子的控制下表达，该启动子可被双链RNA、干扰素或病毒感染诱导。为了评估已建立的转基因小鼠对ADV攻击的抗性，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.