Siciliano Nicholas A, Huang Lan, Eisenlohr Laurence C
Department of Microbiology and Immunology, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, PA, USA.
Methods Mol Biol. 2013;960:219-245. doi: 10.1007/978-1-62703-218-6_18.
The study of antigen processing and presentation is critical to our understanding of the mechanisms that govern immune surveillance. A typical requirement of assays designed to examine antigen processing and presentation is the de novo biosynthesis of a model antigen. Historically, Vaccinia virus (VACV), a poxvirus closely related to Cowpox, has enjoyed widespread use for this purpose. Recombinant poxvirus-based expression has a number of advantages over other systems. Poxviruses accommodate the insertion of large pieces of recombinant DNA into their genome, and recombination and selection are relatively efficient. Poxviruses readily infect a variety of cell types, and they drive rapid and high levels of antigen expression. Additionally, they can be utilized in a variety of assays to study both MHC class I-restricted and MHC class II-restricted antigen processing and presentation. Ultimately, the numerous advantages of poxvirus recombinants have made the Vaccinia expression system a mainstay in the study of processing and presentation over the past two decades. In an attempt to address one shortcoming of VACV while simultaneously retaining the benefits inherent to poxviruses, our laboratory has begun to engineer recombinant Ectromelia viruses. Ectromelia virus, or mousepox, is a natural pathogen of murine cells and performing experiments in the context of a natural host-pathogen relationship may elucidate unknown factors that influence epitope generation and host response. This chapter describes several recombinant poxvirus system protocols used to study both MHC class I and class II antigen processing and presentation, as well as provides insight and troubleshooting techniques to improve the reproducibility and fidelity of these experiments.
对抗原加工与呈递的研究对于我们理解免疫监视机制至关重要。旨在检测抗原加工与呈递的实验的一个典型要求是从头生物合成一种模型抗原。历史上，痘苗病毒（VACV），一种与牛痘密切相关的痘病毒，已广泛用于此目的。基于重组痘病毒的表达相对于其他系统具有许多优势。痘病毒能够将大片段重组DNA插入其基因组，并且重组和筛选相对高效。痘病毒易于感染多种细胞类型，并能驱动抗原快速且高水平表达。此外，它们可用于多种实验，以研究MHC I类限制和MHC II类限制的抗原加工与呈递。最终，痘病毒重组体的众多优势使痘苗表达系统在过去二十年中成为加工与呈递研究的中流砥柱。为了解决VACV的一个缺点，同时保留痘病毒固有的优势，我们实验室已开始构建重组埃可病毒。埃可病毒，即鼠痘，是鼠细胞的天然病原体，在天然宿主 - 病原体关系背景下进行实验可能会阐明影响表位产生和宿主反应的未知因素。本章描述了几种用于研究MHC I类和II类抗原加工与呈递的重组痘病毒系统方案，并提供了有助于提高这些实验的可重复性和准确性的见解及故障排除技术。
