Maclean A R
Division of Virology, IBLS, Institute of Virology, University of Glasgow, Scotland, UK.
Methods Mol Med. 1998;10:19-25. doi: 10.1385/0-89603-347-3:19.
This chapter deals with (1) the preparation of herpes simplex virus (HSV) virion DNA of a quality and purity suitable to be used for the generation of infectious virus, and (2) its use in the preparation of infectious virus. An important development in the understanding of virus genetics and gene products has been the ability to carry out reverse genetics. This is dependent on the ability to manipulate the genome in vitro and reconstitute infectious virus. Our understanding of DNA viruses and positive stranded RNA viruses (where DNA and RNA/cDNA, respectively, are generally infectious) is considerably greater than for negative stranded RNA viruses, where until recently, it had been impossible to generate virus.from either RNA or cDNA. Within the herpesviridae, knowledge of the function of HSV gene products is one of the more advanced owing to the relatively straightforward techniques required to generate virus from HSV-DNA, and to introduce desired mutations by cloning small parts of the genome, manipulating them, and then reintroducing the mutations by a process of cotransfection and in vivo recombination with intact virus DNA. Other α-herpesviruses, such as EHV-1 and PRV, are equally amenable to such manipulation, and knowledge of their gene products is also well advanced. In contrast, this technology is only now, and with much less success, being applied to other members of the family, such as EBV, HCMV, and VZV, and knowledge of their genetics is much less advanced. The use of cosmids to reconstitute intact virus will aid in the advance of knowledge for these viruses. For examples of uses of recombinant DNA technology, the reader is referred to other chapters (especially those on cloning and mutagenesis). I will concentrate on the techniques currently in use in my laboratory, but will also mention other techniques in use elsewhere that may be more appropriate in certain cell types.
（1）制备质量和纯度适合用于产生感染性病毒的单纯疱疹病毒（HSV）病毒粒子DNA；（2）将其用于制备感染性病毒。在病毒遗传学和基因产物理解方面的一项重要进展是能够进行反向遗传学研究。这依赖于在体外操纵基因组并重建感染性病毒的能力。我们对DNA病毒和正链RNA病毒（其中DNA和RNA/cDNA通常具有感染性）的了解比对负链RNA病毒的了解要多得多，直到最近，对于负链RNA病毒，从RNA或cDNA产生病毒一直是不可能的。在疱疹病毒科中，由于从HSV-DNA产生病毒以及通过克隆基因组的小片段、对其进行操作，然后通过共转染和与完整病毒DNA的体内重组过程重新引入突变所需的技术相对简单，HSV基因产物功能的知识是较为先进的之一。其他α疱疹病毒，如EHV-1和PRV，同样适合这种操作，并且它们基因产物的知识也很丰富。相比之下，这项技术直到现在才应用于该家族的其他成员，如EBV、HCMV和VZV，且成功率低得多，对它们遗传学的了解也少得多。使用黏粒重建完整病毒将有助于推进对这些病毒的认识。关于重组DNA技术的应用实例，读者可参考其他章节（特别是关于克隆和诱变的章节）。我将专注于我实验室目前使用的技术，但也会提及其他地方使用的可能在某些细胞类型中更合适的技术。
