Abstract
Background: The 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 UV280nm. Results: The plasmid reconstruct will carry ampr (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 RBD protein. Conclusion: A positive BioVision ELISA test detects <10 pg RBD protein/ml of the sample. A larger sample of the purified RBD protein can be used as a vaccine following a standard formulation and safety protocols. Once administered, the RBD protein will stimulate antibody production against the SARS-CoV-2 virus. The RBD protein has no potential to recombine with human genome.
Highlights
The first human upper respiratory tract infection (URTI) caused by human CoV (H-CoV) was reported in 1965.4-7 The first severe acute respiratory syndrome caused by CoV (SARSCoV) was reported from Guangdong, China, in 2002, that spread over many countries causing an epidemic in the Americas, Europe, and Asia, infecting over 8,098 people and killing about 774 of the infected.[6,8]
SARS-CoV has 99.6% genome sequence homology to CoV found in masked palm civets (Paguma larvata) and 88% – 95% homology to CoV found in several horseshoe bats, Rhinolophus pussilius, R. macrotis, R. pearsoni and R. sinicus.[9,10,11]. This was followed by another outbreak of a CoV epidemic in 2012 that started in Saudi Arabia, which is known as Middle East respiratory syndrome (MERS) and is caused by MERS-CoV.[12,13]
Our proposal We propose to reconstruct an ampr plasmid expression vector carrying receptor-binding domain (RBD) and superfolder green fluorescent protein cDNAs linked by an oligo DNA, coding for a fusion peptide (FP), Asp-Asp-Asp-Asp-Lys.[1]
Summary
A brief history of coronavirus infection Using the “RNA-Dependent RNA Polymerase Molecular Clock”, it was estimated that the common ancestor of coronavirus (CoV) appeared about 10,000 years ago.[2,3] The first human upper respiratory tract infection (URTI) caused by human CoV (H-CoV) was reported in 1965.4-7 The first severe acute respiratory syndrome caused by CoV (SARSCoV) was reported from Guangdong, China, in 2002, that spread over many countries causing an epidemic in the Americas, Europe, and Asia, infecting over 8,098 people and killing about 774 of the infected.[6,8] SARS-CoV has 99.6% genome sequence homology to CoV found in masked palm civets (Paguma larvata) and 88% – 95% homology to CoV found in several horseshoe bats, Rhinolophus pussilius, R. macrotis, R. pearsoni and R. sinicus.[9,10,11]This was followed by another outbreak of a CoV epidemic in 2012 that started in Saudi Arabia, which is known as Middle East respiratory syndrome (MERS) and is caused by MERS-CoV.[12,13] It spread over 27 countries, reaching Western Africa to the west and South Korea to the east, infecting over 2,400 and killing over 850 people.[12,13] The survivors suffered from many diseases including heart, kidney, and multiorgan failures.[12,14,15] In November 2019, another CoV epidemic emerged in Wuhan, China, found to be caused by SARS-CoV-2 infections, leading to a global pandemic. A brief history of coronavirus infection Using the “RNA-Dependent RNA Polymerase Molecular Clock”, it was estimated that the common ancestor of coronavirus (CoV) appeared about 10,000 years ago.[2,3] The first human upper respiratory tract infection (URTI) caused by human CoV (H-CoV) was reported in 1965.4-7 The first severe acute respiratory syndrome caused by CoV (SARSCoV) was reported from Guangdong, China, in 2002, that spread over many countries causing an epidemic in the Americas, Europe, and Asia, infecting over 8,098 people and killing about 774 of the infected.[6,8] SARS-CoV has 99.6% genome sequence homology to CoV found in masked palm civets (Paguma larvata) and 88% – 95% homology to CoV found in several horseshoe bats, Rhinolophus pussilius, R. macrotis, R. pearsoni and R. sinicus.[9,10,11]. 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 stimulate antibody article can be found at the end of the article
Sign-in/Register to view highlights & summary 
Published Version (
Free)
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call