High-Throughput Baculovirus Expression in Insect Cells
High-Throughput Baculovirus Expression in Insect Cells
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DOI：
10.1007/978-1-61779-433-9_33
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发表时间：
2012-01-01
期刊：
影响因子：
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通讯作者：
King, Linda A.
中科院分区：
文献类型：
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作者：
Hitchman, Richard B.;Possee, Robert D.;King, Linda A.
从历史上看，由于所涉及过程的复杂性，传统杆状病毒表达系统很难适应自动化平台。主要瓶颈之一是从亲本病毒中选择重组体。我们开发了一种杆粒载体（flashBAC (TM)），不需要任何形式的选择压力即可将重组病毒与非重组亲本病毒分开。该方法依赖于昆虫细胞中含有目的基因的转移质粒和复制缺陷型杆粒之间的同源重组。感兴趣的基因取代了多角体基因座上的细菌复制子，同时恢复了复制所必需的病毒基因，并且由于只有重组病毒才能复制，因此不需要进一步的分离技术。本章介绍了使用 flashBAC 系统在自动化平台上生产和表达测试多种重组杆状病毒的方法。
Historically, it has been proved difficult to adapt the traditional baculovirus expression systems to an automated platform because of the complexity of the processes involved. One of the major bottlenecks is the selection of recombinant from parental viruses. We have developed a bacmid vector (flashBAC (TM)) that does not require any form of selection pressure to separate recombinant virus from nonrecombinant parental virus. The method relies on homologous recombination in insect cells between a transfer plasmid containing the gene of interest and a replication-deficient bacmid. The gene of interest replaces the bacterial replicon at the polyhedrin locus, simultaneously restoring a virus gene essential for replication, and as only recombinant virus can replicate, no further separation techniques are required. This chapter describes methods for producing and expression testing multiple recombinant baculoviruses on automated platforms using the flashBAC system.