A study protocol to prepare an RBD protein for vaccine against COVID-19 [version 2; peer review: 1 approved with reservations, 1 not approved]

• Published in: F1000 research 2021, Vol.10, p.943
• Main Authors: Jahangir, ZMG Sarwar, Marnik, Arleta Helena
• Format: Article
• Language: English
• ISSN: 2046-1402; 2046-1402
• DOI: 10.12688/f1000research.54738.2

Background: SARS-CoV-2 pandemic is a global threat to humans and the world's economy. Effective and safe vaccines against this virus are essential to control and eradicate the pandemic. The currently applied vaccines carry SARS-CoV-2 spike-protein mRNA/cDNA. These vaccines go through several cellular processes in the recipients for producing antigens. On the contrary, the SARS-CoV-2 RBD (receptor binding domain)-protein is an antigen. It will directly stimulate antibody production against SARS-CoV-2. Hence, we propose to produce SARS-CoV-2 RBD-protein as a fast acting, effective and safe vaccine.  Methods: We propose to reconstruct a plasmid carrying three types of  DNA sequences: RBD cDNA, FP (fusion peptide) DNA and sfGFP(superfolder-green-fluorescent-protein), cDNA creating the RBD-FP-sfGFP DNA within an orf (open-reading-frame). Escherichia coli, C2566H, transformed with the reconstructed plasmid will express RBD-FP-sfGFP fusion protein producing green fluorescent cfu (colony forming unit). The RBD-protein will be separated from the sfGFP using an FP specific enterokinase, and eluted by HIC ( hydrophobic-interaction-chromatography), detected with a BioVision-Elisa-Kit, and quantified by spectrophotometry at UV280 nm and immune simulation will be carried out using C57BL mice.  Results: The plasmid reconstruct will carry amp r (ampicillin-resistant) gene as a selective marker and a T7 promoter controlling the expression of RBD-FP-sfGFP fusion protein. The transformed Escherichia coli will efficiently express the RBD-FP-sfGFP fusion protein. The highly efficient sfGFP fused within the RBD-FP-sfGFP will produce green fluorescent cfu. The RBD-FP-sfGFP protein extract from the green cfu, digested by enterokinase and separated by the HIC will produce pure immunoreactive RBD protein.  Conclusion: A positive BioVision-ELISA test detects