Department of Bioscience, Graduate School of Science and Technology, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka, 422-8529, Japan.
Research Institute of Green Science and Technology, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka, 422-8529, Japan.
Protein Expr Purif. 2022 Feb;190:106010. doi: 10.1016/j.pep.2021.106010. Epub 2021 Nov 2.
Recombinantly expressed VP1 of norovirus self-assembled and formed norovirus-like particles (NoV-LPs). This native VP1 was expressed using the Bombyx mori nucleopolyhedrovirus (BmNPV) expression system in silkworm larva. NoV-LPs were collected from silkworm fat body lysate by density gradient centrifugation. To improve the purity of the NoV-LP, the proteins were further purified using immobilized metal affinity chromatography based on the surface exposed side chain of histidine residues. The additional purification led to a highly purified virus-like particle (VLP). The morphology and size of the purified VLPs were examined using a transmission electron microscope, and dynamic light scattering revealed a monodispersed spherical morphology with a diameter of 34 nm. The purified product had a purity of >90% with a recovery yield of 48.7% (equivalent to 930 μg) from crude lysate, obtained from seven silkworm larvae. In addition, the purified VLP could be recognized by antibodies against GII norovirus in sandwich enzyme-linked immunosorbent assay, which indicated that the silkworm-derived VLP is biologically functional as a NoV-LP in its native state, is structurally correct, and exerts its biological function. Our results suggest that the silkworm-derived NoV-LP may be useful for subsequent applications, such as in a vaccine platform. Moreover, the silkworm-based expression system is known for its robustness, facile up-scalability, and relatively low expense compared to insect cell systems.
诺如病毒的重组 VP1 能够自我组装并形成类诺如病毒样颗粒（NoV-LPs）。这种天然 VP1 是使用家蚕杆状病毒（BmNPV）表达系统在家蚕幼虫中表达的。通过密度梯度离心从蚕脂肪体裂解物中收集 NoV-LPs。为了提高 NoV-LP 的纯度，使用基于组氨酸残基暴露侧链的固定化金属亲和层析进一步对蛋白质进行纯化。额外的纯化导致高度纯化的病毒样颗粒（VLP）。使用透射电子显微镜检查纯化的 VLPs 的形态和大小，动态光散射显示出具有 34nm 直径的单分散球形形态。从七个蚕幼虫的粗裂解物中获得的纯化产物的纯度>90%，回收率为 48.7%（相当于 930μg）。此外，纯化的 VLP 可以被针对 GII 诺如病毒的抗体在夹心酶联免疫吸附测定中识别，这表明蚕源性 VLP 在其天然状态下作为 NoV-LP 具有生物功能，结构正确，并发挥其生物学功能。我们的结果表明，蚕源性 NoV-LP 可能可用于随后的应用，例如疫苗平台。此外，与昆虫细胞系统相比，基于家蚕的表达系统具有稳健性、易于扩大规模和相对较低的成本等优点。
