Hydrophobin-Fused SARS-CoV-2 RBD production in Nicotiana benthamiana L. for COVID-19 serology.

INTRODUCTION: The global health crisis underscores the need for accessible and cost-effective biomedical solutions, especially in resource-limited settings. Plant-based transient expression systems provide a viable method for producing recombinant proteins, such as the SARS-CoV-2 receptor-binding domain (RBD), for diagnostic applications. The addition of hydrophobin enhances protein purification efficiency and cost-effectiveness. METHODS: The study produced recombinant SARS-CoV-2 RBD fused to hydrophobin I (RBD-S-SCV2-HFBI) in Nicotiana benthamiana L. Immunogenicity was evaluated using serum from Swiss mice immunized with viral supernatant. Serum samples from COVID-19 patients were tested for immunogenicity of RBD-S-SCV2-HFBI using SDS-PAGE, Western blotting, and ELISA. Statistical analysis included descriptive statistics and one-way ANOVA with Kruskall-Wallis. RESULTS: The RBD-S-SCV2-HFBI protein was successfully expressed in Nicotiana benthamiana L., demonstrating high immunogenicity. It was significantly recognized by IgG and IgM antibodies in serum samples from COVID-19 patients. The hydrophobin tag improved protein aggregation and purification, enabling efficient and scalable production. CONCLUSION: The study validates the feasibility of using RBD-S-SCV2-HFBI for diagnostic applications. Plant-based systems offer a rapid and cost-effective platform for producing high-quality recombinant proteins, providing scalable solutions to global health emergencies, particularly in resource-limited contexts.