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Abstract：
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.
摘要：
黄病毒病毒样颗粒（VLP）表现出与病毒颗粒惊人的结构相似性，使它们高度适应各种应用，包括疫苗和诊断。因此,增加VLP产量是重要的,并且可以通过优化表达质粒和细胞培养条件来实现。在尝试表达在包膜（E）蛋白中含有G104H突变的基因型III（GIII）日本脑炎病毒（JEV）VLP时，我们未能在COS-1细胞中产生VLP。然而,通过在较低温度下培养质粒转染的细胞来恢复VLP的产生，特别是28°C此外，我们观察到JEVVLP产生的增强与E蛋白中的氨基酸突变无关。在质粒转染的COS-1细胞中产生JEVVLP的最佳条件包括在37°C下初始培养6小时，然后转移到28°C(37/28°C)进行培养。在37/28°C培养条件下，无论其表达是瞬时转染还是克隆选择的细胞，黄病毒VLP的产生在各种哺乳动物细胞系中均显着增加。值得注意的是,克隆选择的表达黄病毒VLP的细胞系始终达到超过1μg/ml的产量。使用单克隆抗体的结合亲和力分析揭示了基因型I（GI）JEV的VLP的相似结合模式，GIIIJEV，西尼罗河病毒(WNV)和在37°C或37/28°C培养条件下产生的登革热病毒血清型2(DENV-2)。总之,我们的研究表明，在37/28°C的培养条件下，黄病毒VLP的产生可以显着提高，而不会影响E蛋白的构象结构。关键词:•低温培养(37/28°C)增强黄病毒VLP的产生。•黄病毒VLP始终实现超过1μg/ml的产量。•37/28°C培养不改变黄病毒VLP的结构。
