Merchlinsky M, Eckert D, Smith E, Zauderer M
Laboratory of Viral Diseases, Food and Drug Administration, Bethesda, Maryland 20892, USA.
Virology. 1997 Nov 24;238(2):444-51. doi: 10.1006/viro.1997.8828.
Poxvirus vectors are extensively used as expression vehicles for protein and antigen expression in eukaryotic cells. Customarily, the foreign DNA is introduced into the poxvirus genome by homologous recombination. An alternative method using direct ligation vectors has been used to efficiently construct chimeric genomes in situations not readily amenable for homologous recombination. We describe the construction and characterization of a new set of direct ligation vectors designed to be universally applicable for the generation of chimeric vaccinia genomes. These vectors contain the pair of unique restriction sites NotI and ApaI to eliminate religation of poxvirus arms and fix the orientation of the insert DNA behind strongly expressing constitutive vaccinia promoters. The insertion cassette has been placed at the beginning of the thymidine kinase gene in vaccinia to use drug selection in the isolation of recombinants. These viruses provide a set of universally applicable direct ligation poxvirus cloning vectors, extending the utility of poxvirus vectors for construction and expression of complex libraries.
痘病毒载体被广泛用作在真核细胞中表达蛋白质和抗原的表达载体。通常，通过同源重组将外源DNA引入痘病毒基因组。在不易进行同源重组的情况下，一种使用直接连接载体的替代方法已被用于高效构建嵌合基因组。我们描述了一组新的直接连接载体的构建和特性，这些载体设计用于普遍适用于嵌合痘苗病毒基因组的产生。这些载体包含一对独特的限制性酶切位点NotI和ApaI，以消除痘病毒臂的重新连接，并将插入DNA的方向固定在强表达的组成型痘苗启动子之后。插入盒已置于痘苗病毒胸苷激酶基因的起始位置，以便在重组体分离中使用药物选择。这些病毒提供了一组普遍适用的直接连接痘病毒克隆载体，扩展了痘病毒载体在构建和表达复杂文库方面的应用。
