Puente-Massaguer Eduard, Gòdia Francesc, Lecina Martí
Departament d'Enginyeria Química, Biològica i Ambiental, Universitat Autònoma de Barcelona, Cerdanyola del Vallès, Barcelona, Spain.
Departament d'Enginyeria Química, Biològica i Ambiental, Universitat Autònoma de Barcelona, Cerdanyola del Vallès, Barcelona, Spain.
J Biotechnol. 2020 Oct 10;322:43-53. doi: 10.1016/j.jbiotec.2020.07.009. Epub 2020 Jul 13.
Insect cells have shown a high versatility to produce multiple recombinant products. The ease of culture, low contamination risk with human pathogens and high expression capacity makes an attractive platform to generate virus-like particles (VLPs). The baculovirus expression vector system (BEVS) has been frequently used to produce these complex nanoparticles. However, the BEVS entails several difficulties in the downstream phase as well as undesirable side-effects due to the expression of baculovirus-derived proteins. In this work, we developed a baculovirus-free system based on polyethylenimine (PEI)-mediated transient gene expression (TGE) of Sf9 cells. An exhaustive study of DNA:PEI polyplex formation was performed and the optimal TGE conditions were determined by the combination of Design of Experiments (DoE) and desirability functions. The TGE approach was successfully applied to produce three model recombinant products with different structural complexities, including eGFP, hSEAP and HIV-1 Gag VLPs. Cell membrane co-localization with the Gag polyprotein was detected by fluorescence microscopy, whereas nanoparticle tracking analysis and flow virometry were applied as high-throughput techniques to monitor the VLP production process. Analysis of VLP production revealed that 48 h after transfection were optimal for VLP harvesting since the ratio of VLPs to extracellular vesicles was the highest. In these conditions, a maximum of 1.9 ± 0.8·10 VLP/mL was achieved, representing a 2.8-fold increase compared to the initial transfection condition. In conclusion, the TGE approach proposed in this study provides a baculovirus-free platform to rapidly produce VLPs and potentially other recombinant products in insect cells.
昆虫细胞已显示出在生产多种重组产品方面具有高度的通用性。其易于培养、感染人类病原体的风险低以及高表达能力，使其成为生产病毒样颗粒（VLP）的有吸引力的平台。杆状病毒表达载体系统（BEVS）已被频繁用于生产这些复杂的纳米颗粒。然而，BEVS在下游阶段存在一些困难，并且由于杆状病毒衍生蛋白的表达会产生不良副作用。在这项工作中，我们基于聚乙烯亚胺（PEI）介导的Sf9细胞瞬时基因表达（TGE）开发了一种无杆状病毒系统。对DNA:PEI多聚体的形成进行了详尽研究，并通过实验设计（DoE）和期望函数的组合确定了最佳TGE条件。TGE方法成功应用于生产三种具有不同结构复杂性的模型重组产品，包括eGFP、hSEAP和HIV-1 Gag VLP。通过荧光显微镜检测细胞膜与Gag多蛋白的共定位，而纳米颗粒跟踪分析和流式病毒测定法作为高通量技术用于监测VLP生产过程。VLP生产分析表明，转染后48小时是收获VLP的最佳时间，因为VLP与细胞外囊泡的比例最高。在这些条件下，最高可达到1.9±0.8·10 VLP/mL，与初始转染条件相比增加了2.8倍。总之，本研究中提出的TGE方法提供了一个无杆状病毒平台，可在昆虫细胞中快速生产VLP以及潜在的其他重组产品。
