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. Previously, many methods required a plaque-assay to separate parental and recombinant virus prior to amplification and use of the recombinant virus. Fortunately, this step is no longer required for most systems currently available. 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 or real-time polymerase chain reaction (PCR). 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
尽管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，其中昆虫细胞用单独的病毒基因（Bac-to-Bac / sup或BaculoDirect鈩 Invitrogen）或病毒基因组和转移载体在后一种情况下，这允许同源重组的天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来创建重组病毒。以前，许多方法需要进行斑块测定以在扩增和使用重组病毒之前分离亲本和重组病毒。幸运的是，目前可用的大多数系统不再需要此步骤历史发展越来越有效的分离重组杆状病毒系统（第3章提供了可用的不同系统和转移载体的全面介绍） 实际细节涵盖：用病毒DNA或病毒DNA和质粒转移载体转染昆虫细胞;一种可用于将重组病毒与必需的亲本（非重组）病毒分离的可靠斑块测定方法;重组病毒小规模扩增方法;并通过斑块测定法或实时聚合酶链反应（PCR） 进行滴定还涵盖了Bac-to-Bac / sup系统独特的方法，包括细菌细胞的转化和分离用于转染昆虫细胞的杆粒DNA
Linda A. King (3); Richard Hitchman (3); Robert D. Possee (4); (5);. Recombinant Baculovirus Isolation. Baculovirus and Insect Cell Expression Protocols[M].DE: Springer, 2016: 73-94