Nakauchi Mina, Kariwa Hiroaki, Kon Yasuhiro, Yoshii Kentaro, Maeda Akihiko, Takashima Ikuo
Laboratory of Public Health, Department of Environmental Veterinary Sciences, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo, Hokkaido, Japan.
Microbiol Immunol. 2008 Dec;52(12):625-30. doi: 10.1111/j.1348-0421.2008.00079.x.
SARS-CoV has four major structural proteins: the N, S, M, and E proteins. To investigate the mechanism of SARS-CoV assembly, we cloned the genes encoding these four proteins into the eukaryotic expression vector pCAGGS and transfected them into 293T cells. When all four expression vectors were co-transfected VLP formed, as confirmed using electron microscopy. Using a rabbit polyclonal antibody specific to the N protein, N-protein-containing particles similar in size to the VLP were also observed by immunoelectron microscopy, indicating that the VLP contained the N protein. Co-immunoprecipitation analyses demonstrated an interaction between the N and M proteins, suggesting that N protein binds directly to M protein to be incorporated into VLP.
严重急性呼吸综合征冠状病毒（SARS-CoV）有四种主要结构蛋白：核衣壳蛋白（N）、刺突蛋白（S）、膜蛋白（M）和包膜蛋白（E）。为了研究SARS-CoV的组装机制，我们将编码这四种蛋白的基因克隆到真核表达载体pCAGGS中，并将其转染到293T细胞中。当共转染所有四种表达载体时，形成了病毒样颗粒（VLP），这通过电子显微镜得以证实。使用针对N蛋白的兔多克隆抗体，通过免疫电子显微镜也观察到了大小与VLP相似的含N蛋白颗粒，表明VLP中含有N蛋白。免疫共沉淀分析证明了N蛋白和M蛋白之间存在相互作用，这表明N蛋白直接与M蛋白结合以被整合到VLP中。
