Jin Hyeop, Cho Ye Rae, Jung Yong Tae
Department of Microbiology, College of Bio-Convergence, Dankook University, Cheonan, 330-714, Korea.
Arch Virol. 2025 Jun 7;170(7):149. doi: 10.1007/s00705-025-06339-w.
SARS-CoV-2 virus-like particles (VLPs) were generated by co-transfecting HEK 293T cells with four plasmids, two for expression of the spike (S) or nucleocapsid (N) protein with a 3xHA tag at the C-terminus, one for expression of the membrane (M) protein, and one for expression of the envelope (E) protein. Co-localization of the S and N proteins within the VLPs was confirmed by Western blot analysis using anti-HA antibodies. To enable fluorescent quantification of VLPs, the C-terminus of the S protein was fused to enhanced green fluorescent protein (EGFP), and the N protein was tagged with EGFP at either the N- or C-terminus. Transient transfection of 293T cells with (S-GFP)NME, S(N-GFP)ME, or S(GFP-N)ME plasmids efficiently produced fluorescent VLPs, each demonstrating the ability to enter A549-hACE2 cells. In addition, a two-plasmid system was developed to simplify fluorescent VLP production by incorporating internal ribosomal entry site elements between N3xHA and S-GFP and between the M and E genes. Transfection of 293T cells with these plasmids produced VLPs with a fourfold higher N protein concentration (800 ng/mL) compared to the four-plasmid system (200 ng/mL), as measured by ELISA. Flow virometry analysis confirmed production of VLPs with an average diameter of 80 nm and a concentration of 1.68 × 10 particles/ml. This study demonstrates that production of fluorescent VLPs using a two-plasmid system is more efficient than the traditional four-plasmid approach. We also demonstrate that flow virometry is a robust method for single-particle analysis of fluorescent VLPs for determining their size and concentration.
通过用四种质粒共转染人胚肾 293T 细胞来产生严重急性呼吸综合征冠状病毒 2（SARS-CoV-2）病毒样颗粒（VLP），其中两种用于表达 C 末端带有 3xHA 标签的刺突（S）蛋白或核衣壳（N）蛋白，一种用于表达膜（M）蛋白，一种用于表达包膜（E）蛋白。使用抗 HA 抗体的蛋白质印迹分析证实了 VLP 内 S 蛋白和 N 蛋白的共定位。为了实现 VLP 的荧光定量，将 S 蛋白的 C 末端与增强型绿色荧光蛋白（EGFP）融合，并将 N 蛋白在 N 末端或 C 末端用 EGFP 标记。用（S-GFP）NME、S（N-GFP）ME 或 S（GFP-N）ME 质粒瞬时转染 293T 细胞可有效产生荧光 VLP，每种都证明有进入 A549-hACE2 细胞的能力。此外，开发了一种双质粒系统，通过在 N3xHA 和 S-GFP 之间以及 M 和 E 基因之间掺入内部核糖体进入位点元件来简化荧光 VLP 的生产。用这些质粒转染 293T 细胞产生的 VLP，通过酶联免疫吸附测定法（ELISA）测量，其 N 蛋白浓度（800 ng/mL）比四质粒系统（200 ng/mL）高四倍。流式病毒测量分析证实产生了平均直径为 80 nm、浓度为 1.68×10颗粒/毫升的 VLP。这项研究表明，使用双质粒系统生产荧光 VLP 比传统的四质粒方法更有效。我们还证明，流式病毒测量是一种用于荧光 VLP 单颗粒分析以确定其大小和浓度的可靠方法。
