Jarvis Donald L
Department of Molecular Biology, University of Wyoming, Laramie, Wyoming, USA.
Methods Enzymol. 2009;463:191-222. doi: 10.1016/S0076-6879(09)63014-7.
In the early 1980s, the first-published reports of baculovirus-mediated foreign gene expression stimulated great interest in the use of baculovirus-insect cell systems for recombinant protein production. Initially, this system appeared to be the first that would be able to provide the high production levels associated with bacterial systems and the eukaryotic protein processing capabilities associated with mammalian systems. Experience and an increased understanding of basic insect cell biology have shown that these early expectations were not completely realistic. Nevertheless, baculovirus-insect cell expression systems have the capacity to produce many recombinant proteins at high levels and they also provide significant eukaryotic protein processing capabilities. Furthermore, important technological advances over the past 20 years have improved upon the original methods developed for the isolation of baculovirus expression vectors, which were inefficient, required at least some specialized expertise and, therefore, induced some frustration among those who used the original baculovirus-insect cell expression system. Today, virtually any investigator with basic molecular biology training can relatively quickly and efficiently isolate a recombinant baculovirus vector and use it to produce their favorite protein in an insect cell culture. This chapter will begin with background information on the basic baculovirus-insect cell expression system and will then focus on recent developments that have greatly facilitated the ability of an average investigator to take advantage of its attributes.
20世纪80年代初,杆状病毒介导的外源基因表达的首次发表报告激发了人们对利用杆状病毒-昆虫细胞系统生产重组蛋白的极大兴趣。最初,这个系统似乎是第一个能够提供与细菌系统相关的高产量以及与哺乳动物系统相关的真核蛋白加工能力的系统。经验以及对基础昆虫细胞生物学的进一步了解表明,这些早期期望并不完全现实。尽管如此,杆状病毒-昆虫细胞表达系统有能力高水平生产多种重组蛋白,并且还具备显著的真核蛋白加工能力。此外,在过去20年里取得的重要技术进步改进了最初为分离杆状病毒表达载体而开发的方法,那些原始方法效率低下,需要至少一些专业知识,因此让使用原始杆状病毒-昆虫细胞表达系统的人感到有些沮丧。如今,几乎任何接受过基础分子生物学培训的研究人员都能够相对快速且高效地分离出重组杆状病毒载体,并利用它在昆虫细胞培养物中生产他们感兴趣的蛋白。本章将首先介绍基本的杆状病毒-昆虫细胞表达系统的背景信息,然后重点讲述最近的一些进展,这些进展极大地便利了普通研究人员利用该系统特性的能力。