Graduate Institute of Microbiology and Public Health, National Chung Hsing University, Taichung, 402, Taiwan.
Institute of Epidemiology and Preventive Medicine, College of Public Health, National Taiwan University, Taipei, 10617, Taiwan.
Appl Microbiol Biotechnol. 2024 Feb 28;108(1):242. doi: 10.1007/s00253-024-13064-y.
Flavivirus virus-like particles (VLPs) exhibit a striking structural resemblance to viral particles, making them highly adaptable for various applications, including vaccines and diagnostics. Consequently, increasing VLPs production is important and can be achieved by optimizing expression plasmids and cell culture conditions. While attempting to express genotype III (GIII) Japanese encephalitis virus (JEV) VLPs containing the G104H mutation in the envelope (E) protein, we failed to generate VLPs in COS-1 cells. However, VLPs production was restored by cultivating plasmid-transfected cells at a lower temperature, specifically 28 °C. Furthermore, we observed that the enhancement in JEV VLPs production was independent of amino acid mutations in the E protein. The optimal condition for JEV VLPs production in plasmid-transfected COS-1 cells consisted of an initial culture at 37 °C for 6 h, followed by a shift to 28 °C (37/28 °C) for cultivation. Under 37/28 °C cultivation conditions, flavivirus VLPs production significantly increased in various mammalian cell lines regardless of whether its expression was transiently transfected or clonally selected cells. Remarkably, clonally selected cell lines expressing flavivirus VLPs consistently achieved yields exceeding 1 μg/ml. Binding affinity analyses using monoclonal antibodies revealed similar binding patterns for VLPs of genotype I (GI) JEV, GIII JEV, West Nile virus (WNV), and dengue virus serotype 2 (DENV-2) produced under both 37 °C or 37/28 °C cultivation conditions. In summary, our study demonstrated that the production of flavivirus VLPs can be significantly improved under 37/28 °C cultivation conditions without affecting the conformational structure of the E protein. KEYPOINTS: • Low-temperature culture (37/28 °C) enhances production of flavivirus VLPs. • Flavivirus VLPs consistently achieved yields exceeding 1 μg/ml. • 37/28 °C cultivation did not alter the structure of flavivirus VLPs.
黄病毒病毒样颗粒 (VLPs) 表现出与病毒颗粒惊人的结构相似性，因此非常适合各种应用，包括疫苗和诊断。因此，提高 VLPs 的产量非常重要，可以通过优化表达质粒和细胞培养条件来实现。在尝试表达含有包膜 (E) 蛋白 G104H 突变的基因型 III (GIII) 日本脑炎病毒 (JEV) VLPs 时，我们未能在 COS-1 细胞中产生 VLPs。然而，通过在较低温度（具体为 28°C）培养转染质粒的细胞，VLPs 的产生得到了恢复。此外，我们观察到 JEV VLPs 产量的提高与 E 蛋白中的氨基酸突变无关。在转染质粒的 COS-1 细胞中生产 JEV VLPs 的最佳条件是在初始培养时于 37°C 培养 6 小时，然后转移到 28°C（37/28°C）培养。在 37/28°C 培养条件下，各种哺乳动物细胞系中黄病毒 VLPs 的产量均显著增加，无论其表达是瞬时转染还是克隆选择的细胞。值得注意的是，克隆选择的表达黄病毒 VLPs 的细胞系的产量始终超过 1μg/ml。使用单克隆抗体进行的结合亲和力分析表明，在 37°C 或 37/28°C 培养条件下产生的基因型 I (GI) JEV、GIII JEV、西尼罗河病毒 (WNV) 和登革热病毒血清型 2 (DENV-2) 的 VLPs 具有相似的结合模式。总之，我们的研究表明，在不影响 E 蛋白构象结构的情况下，37/28°C 培养条件下黄病毒 VLPs 的产量可以显著提高。关键点：• 低温培养（37/28°C）可增强黄病毒 VLPs 的产量。• 黄病毒 VLPs 的产量始终超过 1μg/ml。• 37/28°C 培养不会改变黄病毒 VLPs 的结构。
