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 ± 0.8·10⁹ 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.

昆虫细胞显示出产生多种重组产物的高度通用性。培养的简便性，人类病原体的低污染风险和高表达能力使它成为产生病毒样颗粒（VLP）的诱人平台。杆状病毒表达载体系统（BEVS）已经常用于生产这些复杂的纳米颗粒。然而，由于杆状病毒衍生的蛋白的表达，BEVS在下游阶段存在一些困难以及不良的副作用。在这项工作中，我们开发了一种基于聚乙烯亚胺（PEI）介导的Sf9细胞瞬时基因表达（TGE）的无杆状病毒的系统。进行了详尽的DNA：PEI复合物形成研究，并通过实验设计（DoE）和期望功能的组合确定了最佳TGE条件。TGE方法已成功应用于生产三种结构复杂程度不同的模型重组产品，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分析则被用作高通量技术来监测VLP生产过程。对VLP产生的分析表明，转染后48小时最适合VLP收获，因为VLP与细胞外囊泡的比例最高。在这些条件下，最大值为1.9±0.8·10 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分析则被用作高通量技术来监测VLP生产过程。对VLP产生的分析表明，转染后48小时最适合VLP收获，因为VLP与细胞外囊泡的比例最高。在这些条件下，最大值为1.9±0.8·10 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分析则被用作高通量技术来监测VLP生产过程。对VLP产生的分析表明，转染后48小时最适合VLP收获，因为VLP与细胞外囊泡的比例最高。在这些条件下，最大值为1.9±0.8·10达到⁹ VLP / mL，比初始转染条件提高了2.8倍。总之，本研究中提出的TGE方法提供了无杆状病毒的平台，可在昆虫细胞中快速产生VLP和可能的其他重组产物。