摘要
Although there are several different methods available of making recombinant baculovirus expression vectors (reviewed in Chapter 3), all require a stage in which insect cells are transfected with either the virus genome alone (Bac-to-Bac® or BaculoDirect™, Invitrogen) or virus genome and transfer vector. In the latter case, this allows the natural process of homologous recombination to transfer the foreign gene, under control of the polyhedrin or other baculovirus gene promoter, from the transfer vector to the virus genome to create the recombinant virus. Additionally, many systems require a plaque-assay to separate parental and recombinant virus prior to amplification and use of the recombinant virus. This chapter provides an overview of the historical development of increasingly more efficient systems for the isolation of recombinant baculoviruses (Chapter 3 provides a full account of the different systems and transfer vectors available). The practical details cover: transfection of insect cells with either virus DNA or virus DNA and plasmid transfer vector; a reliable plaque-assay method that can be used to separate recombinant virus from parental (nonrecombinant) virus where this is necessary; methods for the small-scale amplification of recombinant virus; and subsequent titration by plaque-assay. Methods unique to the Bac-to-Bac system are also covered and include the transformation of bacterial cells and isolation of bacmid DNA ready for transfection of insect cells.
摘要译文
虽然有几种不同的方法可用于制备重组杆状病毒表达载体（见第3章），所有这些都需要一个阶段，用单独的病毒基因组（Bac-to-Bac®或BaculoDirect™，Invitrogen）或病毒基因组和转移载体转染昆虫细胞。在后一种情况下，这允许同源重组的自然过程在多角体蛋白或其他杆状病毒基因启动子的控制下转移外源基因，从转移载体到病毒基因组产生重组病毒。另外，许多系统在扩增和使用重组病毒之前需要噬斑测定来分离亲本和重组病毒。用于分离重组杆状病毒的越来越有效的系统的历史发展（第3章提供了可用的不同系统和转移载体的完整说明）。实际细节包括：用病毒DNA或病毒DNA和质粒转移载体转染昆虫细胞;一种可靠的噬斑测定方法，可用于在必要时将重组病毒与亲本（非重组）病毒分离;小规模扩增重组病毒的方法;通过噬斑测定随后滴定。还涵盖了Bac-to-Bac系统独有的方法，包括细菌细胞的转化和bacmid DNA的分离，准备转染昆虫细胞。
Linda A. King[1];Richard Hitchman[1];Robert D. Possee[2]. Recombinant Baculovirus Isolation. Baculovirus and Insect Cell Expression Protocols[M].DE: Springer, 2007: 77-93