From In Silico to In Vivo: Characterizing Ag-RBDN331-V524 for Effective COVID-19 Vaccine Development

COVID-19 has become a challenge of the century to the healthcare system. One of the best targets to produce the COVID-19 vaccine is the receptor binding domain (RBD) which is located in the Spike protein of Coronavirus. This domain is extremely conserved among different variants of COVID-19. In this study, the most potent region of RBD was selected to design a vaccine against COVID-19 in E. coli as an expression system. The shuttle vector was applied to express the vaccine construct in E. coli . The primary vector is a backbone plasmid pBluescriptIISK (+) and a Tn5 transposon is a secondary vector of 1657 bp inside. The C-phycocyanin operon including the gene cassette was embedded in Tn5. The quantitation of total protein was done by Bradford assay. Then, SDS-PAGE and Western Blot were carried out to characterize and confirm recombinant protein expression. Affinity chromatography was performed for the purification of recombinant protein. The molecular weight of the RBD protein was 34 kDa which is compatible with western blot results. The aim of this study was expression of RBD domain in E. coli which could apply in a future study to the production of vaccine against COVID-19 based on a host that has ideal C-phycocyanin expression. The selected RBD sequence has a complete identity to the newest variant. The short length of the sequence selected in this study leads to increased solubility and decreased allergenicity. On the contrary, this trait has led to a decrease in the probability of mutation, which can cover new variants of this virus.