Abstract：
The insect cell-baculovirus expression vector (IC-BEV) platform has enabled small research-scale and large commercial-scale production of recombinant proteins and therapeutic biologics including recombinant adeno-associated virus (rAAV)-based gene delivery vectors. The wide use of this platform is comparable with other mammalian cell line-based platforms due to its simplicity, high-yield, comparable quality attributes, and robust bioprocessing features. In this chapter, we describe a rAAV production protocol employing one of the recent modifications of the One-Bac platform that consists of a stable transformed Sf9 cell line carrying AAV Rep2/Cap5 genes that are induced upon infection with a single recombinant baculovirus expression vector harboring the transgene of interest (rAAV genome). The overall protocol consists of essential steps including rBEV working stock preparation, rAAV production, and centrifugation-based clarification of cell culture lysate. The same protocol can also be applied for rAAV vector production using traditional Three-Bac, Two-Bac, and Mono-Bac platforms without requiring significant changes.
摘要：
昆虫细胞-杆状病毒表达载体(IC-BEV)平台使得能够进行小规模研究和大规模商业生产重组蛋白和治疗性生物制品,包括基于重组腺相关病毒(rAAV)的基因递送载体。由于其简单性，该平台的广泛使用可与其他基于哺乳动物细胞系的平台相媲美。高产,可比较的质量属性，和强大的生物处理功能。在这一章中,我们描述了使用One-Bac平台的最新修改之一的rAAV生产方案,其由携带AAVRep2/Cap5基因的稳定转化的Sf9细胞系组成,所述AAVRep2/Cap5基因在感染后被单个重组杆状病毒表达载体诱导,所述载体含有感兴趣的转基因(rAAV基因组)。总体方案包括基本步骤，包括rBEV工作库存准备，rAAV生产,和基于离心的细胞培养裂解物的澄清。相同的协议也可以应用于使用传统的Three-Bac的rAAV载体生产，两个Bac,和Mono-Bac平台，无需进行重大更改。
