Abstract：
Baculoviruses are widely used for their potential as biological pesticide and as platform for the production of recombinant proteins and gene therapy vectors. The Baculovirus Expression Vector System (BEVS) is used for high level of expression of (multiple) proteins in insect cells. Baculovirus recombinants can be quickly constructed by transposition of the gene(s) of interest into a so-called bacmid, which is a baculovirus infectious clone maintained as single-copy, bacterial artificial chromosome in Escherichia coli. A two-step homologous recombineering technique using the lambda-red system in E. coli allows for scarless editing of the bacmid with PCR products based on sequence homology. In the first step, a selection cassette with 50 bp homology arms, typically generated by PCR, is inserted into the designated locus. In the second step, the selection cassette is removed based on a negative selection marker, such as SacB or rpsL. This lambda-red recombineering technique can be used for multiple gene editing purposes, including (large) deletions, insertions, and even single point mutations. Moreover, since there are no remnants of the editing process, successive modifications of the same bacmid are possible. This chapter provides detailed instructions to design and perform two-step homologous recombineering of baculovirus bacmid DNA in E. coli. We present two case studies demonstrating the utility of this technique for creating a deletion mutant of the chitinase and cathepsin genes and for introducing a single point mutation in the baculovirus gene gp41. This scarless genome editing approach can facilitate functional studies of baculovirus genes and improve the production of recombinant proteins using the BEVS.
摘要：
杆状病毒因其作为生物农药的潜力以及作为生产重组蛋白和基因治疗载体的平台而被广泛使用。杆状病毒表达载体系统(BEVS)用于在昆虫细胞中高水平表达(多种)蛋白质。杆状病毒重组体可以通过将感兴趣的基因转座到所谓的bacmid中快速构建。这是一种单拷贝的杆状病毒感染性克隆，大肠杆菌中的细菌人工染色体。在大肠杆菌中使用λ-red系统的两步同源重组工程技术允许使用基于序列同源性的PCR产物对杆粒进行无疤痕编辑。第一步,具有50bp同源臂的选择盒，通常通过PCR产生，插入到指定的基因座中。第二步,选择盒基于阴性选择标记被移除，例如SacB或rpsL。这种lambda-red重组工程技术可用于多种基因编辑目的，包括(大)删除，插入,甚至单点突变。此外，由于没有编辑过程的残余，相同的bacmid的连续修改是可能的。本章提供了在大肠杆菌中设计和执行杆状病毒杆粒DNA两步同源重组的详细说明。我们提供了两个案例研究，证明了该技术可用于创建几丁质酶和组织蛋白酶基因的缺失突变体以及在杆状病毒基因gp41中引入单点突变。这种无疤痕的基因组编辑方法可以促进杆状病毒基因的功能研究，并使用BEVS改善重组蛋白的生产。
