Expression of the RBD Antigen Fused T4 Peptide and Human C5a Fragments of SARS-CoV-2 in Nicotiana benthamiana

This study investigated the expression of a novel fusion peptide, Kozak-A3Dsp-RBD-T4-Linker-C5a-SEKDEL, in Nicotiana benthamiana plants, exploring its potential as a SARS-CoV-2 vaccine candidate. The 1392 bp sequence, encoding the SARS-CoV-2 RBD, a T4 peptide from the S2 subunit, and human C5a, was successfully cloned into the pMYV719 plant expression vector. This construct was then conjugated into Agrobacterium tumefaciens LBA4404 for transient expression in N. benthamiana leaves via infiltration. The p19 gene was co-infiltrated to enhance peptide expression by inhibiting gene silencing. Successful infiltration was confirmed by PCR, showing a 1.4 kb DNA band consistent with the target sequence. Protein expression was subsequently analyzed by SDS-PAGE and western blot. A prominent protein band of approximately 55 kDa was observed, slightly larger than the theoretical 51.5 kDa, likely due to post-translational modifications common in plant systems. Western blot analysis confirmed specific binding of the antibody to the RBD-containing peptide. Expression levels varied across time points, with the highest signal intensity observed at 6 days post-infiltration. This finding aligns with established transient expression patterns in N. benthamiana. This study demonstrates the successful expression of a multi-component SARS-CoV-2 antigen in N. benthamiana, highlighting its potential for plant-based vaccine production.