Vasques Raquel Medeiros, Lacorte Cristiano, da Luz Leonardo Lopes, Aranda Miguel A, Nagata Tatsuya
Departamento de Biologia Celular, Universidade de Brasília, Campus Darcy Ribeiro, Brasília, DF, 70910-900, Brazil.
Embrapa Recursos Genéticos e Biotecnologia, Brasília, DF, 70297-400, Brazil.
Mol Biol Rep. 2019 Feb;46(1):97-103. doi: 10.1007/s11033-018-4449-4. Epub 2018 Oct 26.
Plants are becoming an interesting alternative system for the heterologous production of pharmaceutical proteins, providing a more scalable, cost-effective, and biologically safer option than the current expression systems. The development of plant virus expression vectors has allowed rapid and high-level transient expression of recombinant genes, and, in turn, provided an attractive plant-based production platform. Here we report the development of vectors based on the tobamovirus Pepper mild mottle virus (PMMoV) to be used in transient expression of foreign genes. In this PMMoV vector, a middle part of the viral coat protein gene was replaced by the green fluorescent protein (GFP) gene, and this recombinant genome was assembled in a binary vector suitable for plant agroinoculation. The accumulation of GFP was evaluated by observation of green fluorescent signals under UV light and by western blotting. Furthermore, by using this vector, the multiepitope gene for chikungunya virus was successfully expressed and confirmed by western blotting. This PMMoV-based vector represents an alternative system for a high-level production of heterologous protein in plants.
植物正成为用于异源生产药用蛋白质的一种有趣的替代系统，与当前的表达系统相比，它提供了一种更具扩展性、成本效益更高且生物安全性更高的选择。植物病毒表达载体的发展使得重组基因能够快速、高水平地瞬时表达，进而提供了一个有吸引力的基于植物的生产平台。在此，我们报告基于烟草花叶病毒属辣椒轻斑驳病毒（PMMoV）的载体的开发，该载体用于外源基因的瞬时表达。在这个PMMoV载体中，病毒外壳蛋白基因的中间部分被绿色荧光蛋白（GFP）基因取代，并且这个重组基因组被组装在一个适合植物农杆菌接种的二元载体中。通过在紫外光下观察绿色荧光信号以及蛋白质免疫印迹法来评估GFP的积累情况。此外，通过使用这个载体，基孔肯雅病毒的多表位基因得以成功表达，并通过蛋白质免疫印迹法得到证实。这种基于PMMoV的载体代表了一种在植物中高水平生产异源蛋白质的替代系统。
