摘要
A method is described for the highly efficient recovery of recombinant pseudorabies virions; the approach should be applicable to other herpesviruses. Pseudorabies virus (PRV) strain PRV509 contains a unique EcoRI site in its genome, largely replacing the glycoprotein gC gene. By digesting PRV509 DNA with EcoRI prior to cotransfection with plasmid DNA that harbored a cloned copy of gC, we isolated recombinant viruses containing the cloned gC allele at a frequency exceeding 75%. This represented up to a 37-fold increase over the use of intact viral DNA in cotransfection experiments, and may eliminate the need for phenotypic screening of recombinants. Closer analysis of the recombinant viruses revealed that genetic markers up to 1 kilobase pair apart could be recombined into the genome using the EcoRI-digested DNA. Overall, the increased frequency of recombinant viruses can be explained if homologous recombination at sites of double-strand breakage is a more efficient repair mechanism than the re-annealing and ligation of the break itself.
摘要译文
描述了高效回收重组伪狂犬病毒粒子的方法;该方法应适用于其他疱疹病毒。伪狂犬病病毒（PRV）PRV509在其基因组中含有一个独特的EcoRI位点，在很大程度上取代了糖蛋白gC基因。通过用EcoRI消化PRV509 DNA，然后与包含gC克隆拷贝的质粒DNA共转染，我们分离出含有克隆的gC等位基因的重组病毒，其频率超过75％。这比共转染实验中完整病毒DNA的使用增加了37倍，并且可以消除对重组体的表型筛选的需要。对重组病毒的更接近的分析显示，使用EcoRI消化的DNA可以将多至1千碱基对的遗传标记重组到基因组中。总体，如果在双链断裂位点的同源重组是更有效的修复机制而不是重新退火和连接断裂本身，则可以解释重组病毒的突变。
PatrickRyan;Frank L.Shankly;. A double-strand break in a herpesvirus genome stimulates targeted homologous recombination with exogenous, cloned viral sequences[J]. Journal of Virological Methods, 1996,57(1): 95-107