Mäkelä Anna R, Ernst Wolfgang, Grabherr Reingard, Oker-Blom Christian
Cold Spring Harb Protoc. 2010 Mar;2010(3):pdb.prot5396. doi: 10.1101/pdb.prot5396.
The baculovirus expression vector system has been used extensively to produce numerous proteins originating from both prokaryotic and eukaryotic sources. In addition to easy cloning techniques and abundant viral propagation, the system's insect cell environment provides eukaryotic post-translational modification machinery. The baculovirus display vector system provides a number of advantages over prokaryotic systems, allowing the combination of genotype with phenotype, enabling presentation of foreign peptides or even complex proteins on the baculoviral envelope or capsid. Baculoviruses permit larger gene insertions, are easily propagated, and can be grown to high titers. Furthermore, surface modifications of the viral capsid enable specific targeting. This strategy can be used to enhance viral binding and entry to a wide variety of both dividing and nondividing mammalian cells, as well as to produce antibodies against the displayed antigen. In addition, the technology should enable modifications of intracellular behavior, i.e., trafficking of recombinant "nanoparticles," a highly relevant feature for studies of targeted gene or protein delivery. After generating the display viral stock, it is important to confirm the presence and functionality of the displayed peptides or proteins on the viral particles before proceeding to further experiments. Accordingly, infected insect cells and budded virions can be analyzed by a variety of methods using appropriate antibodies. This protocol describes a standard immunoelectron microscopy technique in detail.
杆状病毒表达载体系统已被广泛用于生产源自原核和真核来源的多种蛋白质。除了简便的克隆技术和丰富的病毒繁殖外，该系统的昆虫细胞环境还提供真核翻译后修饰机制。杆状病毒展示载体系统相对于原核系统具有许多优势，它允许基因型与表型相结合，能够在杆状病毒包膜或衣壳上展示外源肽甚至复杂蛋白质。杆状病毒允许插入更大的基因，易于繁殖，并且可以生长到高滴度。此外，病毒衣壳的表面修饰能够实现特异性靶向。这种策略可用于增强病毒与多种分裂和非分裂哺乳动物细胞的结合与进入，以及产生针对所展示抗原的抗体。此外，该技术应能够修饰细胞内行为，即重组“纳米颗粒”的运输，这是靶向基因或蛋白质递送研究的一个高度相关特征。在制备展示病毒储备液后，在进行进一步实验之前，确认病毒颗粒上所展示肽或蛋白质的存在和功能非常重要。因此，可以使用适当的抗体通过多种方法分析感染的昆虫细胞和出芽的病毒粒子。本方案详细描述了一种标准的免疫电子显微镜技术。
