Center for Infectious Diseases and Vaccinology, Biodesign Institute, Arizona State University, Tempe, Arizona, USA.
Biotechniques. 2011 May;50(5):303-9. doi: 10.2144/000113667.
Vaccinia virus has been a powerful tool in molecular biology and vaccine development. The relative ease of inserting and expressing foreign genes combined with its broad host range has made it an attractive antigen delivery system against many heterologous diseases. Many different approaches have been developed to isolate recombinant vaccinia virus generated from homologous recombination; however, most are time-consuming, often requiring a series of passages or specific cell lines. Herein we introduce a rapid method for isolating recombinants using the antibiotic coumermycin and the interferon-associated PKR pathway to select for vaccinia virus recombinants. This method uses a negative selection marker in the form of a fusion protein, GyrB-PKR, consisting of the coumermycin dimerization domain of Escherichia coli gyrase subunit B fused to the catalytic domain of human PKR. Coumermycin-dependent dimerization of this protein results in activation of PKR and the phosphorylation of translation initiation factor, eIF2α. Phosphorylation of this factor leads to an inhibition of protein synthesis, and an inhibition of virus replication. In the presence of coumermycin, recombinants are isolated due to the loss of this coumermycin-sensitive gene by homologous recombination. We demonstrate that this method of selection is highly efficient and requires limited rounds of enrichment to isolate recombinant virus.
牛痘病毒在分子生物学和疫苗开发中是一种强大的工具。它插入和表达外源基因相对容易，宿主范围广泛，因此成为针对许多异源疾病的有吸引力的抗原传递系统。已经开发出许多不同的方法来分离同源重组产生的重组牛痘病毒；然而，大多数方法耗时耗力，通常需要一系列传代或特定的细胞系。在此，我们介绍了一种使用抗生素氯霉素和干扰素相关的 PKR 途径选择牛痘病毒重组体的快速方法。该方法使用融合蛋白 GyrB-PKR 的负选择标记，该融合蛋白由大肠杆菌拓扑异构酶 B 亚基的氯霉素二聚化结构域与人类 PKR 的催化结构域融合而成。该蛋白的氯霉素依赖性二聚化导致 PKR 的激活和翻译起始因子 eIF2α 的磷酸化。该因子的磷酸化导致蛋白质合成的抑制和病毒复制的抑制。在氯霉素存在的情况下，由于同源重组导致这种氯霉素敏感基因的丢失，因此可以分离重组体。我们证明这种选择方法非常有效，仅需有限轮次的富集即可分离重组病毒。
