Mairhofer Juergen, Lara Alvaro R
Department of Biotechnology, University of Natural Resources and Life Sciences, Vienna, Austria.
Methods Mol Biol. 2014;1139:505-41. doi: 10.1007/978-1-4939-0345-0_38.
Recent developments in DNA vaccine research provide a new momentum for this rather young and potentially disruptive technology. Gene-based vaccines are capable of eliciting protective immunity in humans to persistent intracellular pathogens, such as HIV, malaria, and tuberculosis, for which the conventional vaccine technologies have failed so far. The recent identification and characterization of genes coding for tumor antigens has stimulated the development of DNA-based antigen-specific cancer vaccines. Although most academic researchers consider the production of reasonable amounts of plasmid DNA (pDNA) for immunological studies relatively easy to solve, problems often arise during this first phase of production. In this chapter we review the current state of the art of pDNA production at small (shake flasks) and mid-scales (lab-scale bioreactor fermentations) and address new trends in vector design and strain engineering. We will guide the reader through the different stages of process design starting from choosing the most appropriate plasmid backbone, choosing the right Escherichia coli (E. coli) strain for production, and cultivation media and scale-up issues. In addition, we will address some points concerning the safety and potency of the produced plasmids, with special focus on producing antibiotic resistance-free plasmids. The main goal of this chapter is to make immunologists aware of the fact that production of the pDNA vaccine has to be performed with as much as attention and care as the rest of their research.
DNA疫苗研究的最新进展为这项尚处起步阶段且具有潜在颠覆性的技术注入了新的活力。基于基因的疫苗能够在人体中引发针对持续性细胞内病原体（如HIV、疟疾和结核病）的保护性免疫，而传统疫苗技术迄今在这些病原体上均告失败。最近对编码肿瘤抗原的基因的鉴定和表征推动了基于DNA的抗原特异性癌症疫苗的发展。尽管大多数学术研究人员认为，为免疫学研究生产适量的质粒DNA（pDNA）相对容易解决，但在生产的第一阶段往往会出现问题。在本章中，我们回顾了小规模（摇瓶）和中规模（实验室规模生物反应器发酵）pDNA生产的当前技术水平，并探讨了载体设计和菌株工程的新趋势。我们将引导读者了解工艺设计的不同阶段，从选择最合适的质粒骨架、选择合适的大肠杆菌（E. coli）生产菌株、培养介质以及放大问题等方面入手。此外，我们将讨论一些有关所生产质粒的安全性和效力的问题，特别关注生产无抗生素抗性的质粒。本章的主要目标是让免疫学家认识到，生产pDNA疫苗必须像他们研究的其他方面一样，给予同样多的关注和谨慎。
