We have investigated the use of transient expression to produce virus-like particles (VLPs) of severe acute respiratory syndrome coronavirus 2, the causative agent of COVID-19, in Nicotiana benthamiana. Expression of a native form of the spike (S) protein, either alone or in combination with the envelope (E) and membrane (M) proteins, all of which were directed to the plant membranes via their native sequences, was assessed. The full-length S protein, together with degradation products, could be detected in total protein extracts from infiltrated leaves in both cases. Particles with a characteristic ‘crown-shaped’ or ‘spiky’ structure could be purified by density gradient centrifugation. Enzyme-linked immunosorbent assays using anti-S antibodies showed that threefold higher levels of VLPs containing the full-length S protein were obtained by infiltration with S alone, compared to co-infiltration of S with M and E. The S protein within the VLPs could be cleaved by furin in vitro and the particles showed reactivity with serum from recovering COVID-19 patients, but not with human serum taken before the pandemic. These studies show that the native S protein expressed in plants has biological properties similar to those of the parent virus. We show that the approach undertaken is suitable for the production of VLPs from emerging strains and we anticipate that the material will be suitable for functional studies of the S protein, including the assessment of the effects of specific mutations. As the plant-made material is noninfectious, it does not have to be handled under conditions of high containment.

中文翻译：

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呈现天然刺突蛋白的 SARS-CoV-2 病毒样颗粒的植物表达和表征


我们研究了利用瞬时表达在本塞姆氏烟草中产生严重急性呼吸综合征冠状病毒 2（COVID-19 的病原体）的病毒样颗粒 (VLP)。评估了刺突 (S) 蛋白的天然形式的表达，无论是单独的还是与包膜 (E) 和膜 (M) 蛋白组合，所有这些蛋白都通过其天然序列定向到植物膜。在这两种情况下，在渗透叶子的总蛋白提取物中都可以检测到全长 S 蛋白以及降解产物。具有特征性“冠形”或“尖刺”结构的颗粒可以通过密度梯度离心进行纯化。使用抗 S 抗体的酶联免疫吸附测定表明，与 S 与 M 和 E 共同浸润相比，单独用 S 浸润获得的含有全长 S 蛋白的 VLP 水平高出三倍。VLP 内的 S 蛋白在体外可以被弗林蛋白酶裂解，并且这些颗粒与康复的 COVID-19 患者的血清有反应性，但与大流行前采集的人血清没有反应性。这些研究表明，植物中表达的天然S蛋白具有与亲本病毒相似的生物学特性。我们表明所采用的方法适用于从新兴菌株中生产 VLP，并且我们预计该材料将适用于 S 蛋白的功能研究，包括评估特定突变的影响。由于植物材料不具有传染性，因此不必在高度封闭的条件下进行处理。