Nagel Claus-Henning, Pohlmann Anja, Sodeik Beate
Heinrich Pette Institute-Leibniz-Institute for Experimental Virology, Hamburg, Germany.
Methods Mol Biol. 2014;1144:43-62. doi: 10.1007/978-1-4939-0428-0_4.
Bacterial artificial chromosomes (BACs) are suitable vectors not only to maintain the large genomes of herpesviruses in Escherichia coli but also to enable the traceless introduction of any mutation using modern tools of bacterial genetics. To clone a herpes simplex virus genome, a BAC replication origin is first introduced into the viral genome by homologous recombination in eukaryotic host cells. As part of their nuclear replication cycle, genomes of herpesviruses circularize and these replication intermediates are then used to transform bacteria. After cloning, the integrity of the recombinant viral genomes is confirmed by restriction length polymorphism analysis and sequencing. The BACs may then be used to design virus mutants. Upon transfection into eukaryotic cells new herpesvirus strains harboring the desired mutations can be recovered and used for experiments in cultured cells as well as in animal infection models.
细菌人工染色体（BACs）是合适的载体，不仅可用于在大肠杆菌中维持疱疹病毒的大基因组，还能利用现代细菌遗传学工具无痕引入任何突变。为克隆单纯疱疹病毒基因组，首先通过真核宿主细胞中的同源重组将BAC复制起点引入病毒基因组。作为其核复制周期的一部分，疱疹病毒基因组会环化，然后这些复制中间体用于转化细菌。克隆后，通过限制性片段长度多态性分析和测序确认重组病毒基因组的完整性。然后，BACs可用于设计病毒突变体。转染到真核细胞后，可回收携带所需突变的新疱疹病毒株，并用于培养细胞实验以及动物感染模型实验。
