Expression and immunoassay assessment of immunomodulatory PanDR and SARS-CoV-2 receptor-binding domain antigen in...

The gap in technical capacity and inadequate infrastructure for biomanufacturing limits progress in vaccine development and biomanufacturing in low- and middle-income countries (LMICs). Peptide subunit antigens offer a promising solution to producing cost-effective, safe and protective vaccines for SARS-CoV-2 and other emerging viral diseases. The receptor-binding domain (RBD) of the SARS-CoV-2 spike protein, which mediates viral entry through interaction with the human angiotensin-converting enzyme 2 (ACE2) receptor, represents a suitable model antigen for building and strengthening local capacity. The aim of this study is to express and validate a recombinant SARS-CoV-2 RBD protein as a vaccine candidate using bacterial expression systems. In this study the RBD sequence is linked to PanDR epitope sequence, an immunostimulant' then codon optimized for bacterial expression. The physicochemical parameter of the protein sequence was assessed using in-silico tools to assess its suitability. The sequence was synthesized chemically, and cloned into a pMAL-c6T expression vector, then transformed into Escherichia coli BL21-DE3 cells. The protein was expressed by IPTG induction for 3 h in Luria-Bertani nutrient media and then purified using affinity chromatography. The recombinant protein was successfully expressed in a bacterial expression system as revealed by a distinct band at the expected molecular weight of approximately 74.5 kDa, on SDS-PAGE corresponding to the MBP-RBD fusion protein. Western blot analysis further confirmed the identity of the protein through specific recognition by anti-MBP antibody. The results show that the modified receptor binding domain protein of SARS-CoV-2 was successfully synthesized, cloned, expressed and validated for further immunological evaluation.