Part:BBa_K4861000
wuhan-SARS-CoV2 RBD
The spike protein of COVID-19 is a key protein for infecting the human body. It enters host cells by binding to the ACE2 receptor on the surface of the host cells. Therefore, the S protein is the preferred choice for designing recombinant protein vaccines against the novel coronavirus. The S protein is divided into two subunits, S1 and S2, with S1 containing a receptor-binding domain (RBD), which is a critical structure for the virus to enter host cells.
wuhan-SARS-CoV2 RBD
Properties
- Name: wuhan-SARS-CoV2 RBD
- Base Pairs: 793 bp
- Origin: the S protein from the Wuhan strain (SARS-CoV-2)
- Properties: receptor-binding domain
Usage and Biology
New coronavirus (SARS-CoV-2) is a single-stranded, positive-stranded RNA virus belonging to the Coronaviridae family, one of the human pathogenic coronaviruses with an RNA genome of approximately 30,000 nucleotides encoding a number of structural and nonstructural proteins of which the membrane, envelope, and spikelike proteins are the major proteins that make up the virus.
COVID-19 infection is mainly transmitted through the respiratory tract, and humans can be infected by inhaling virus particles. Virus particles can be transmitted through the air, as well as through contact with infected individuals or objects. After COVID-19 infects the human body, it mainly binds to the ACE2 receptor on the surface of the host cell through spike protein (S protein), and then enters the host cell for replication and infection. In this process, there are multiple biological mechanisms leading to human injury.
The S (spike) protein of COVID-19 is the key protein for it to infect the human body. It mediates the virus to enter the host cell by binding to the ACE2 receptor on the surface of the host cell. Therefore, S protein is the preferred choice for designing a recombinant protein vaccine against COVID-19. The S protein is divided into two subunits, S1 and S2, where the S1 subunit contains a receptor binding domain (RBD), which is a key structure for viruses entering host cells.
Cultivation, Purification and SDS-PAGE
In the initial phase, we began with extracting the pET28a plasmid, which served as the foundational framework. Following this, we conducted double enzyme cuts on both the blank plasmid and the gene segments of viral strain – Wuhan – using NcoI and XhoI enzymes. After these cuts, we merged the target fragments from four viral strains with the pET28a plasmid using T4 DNA ligase. This intricate process transported the target fragments onto the blank plasmid.
Subsequently, we transferred the constructed plasmids into DH5α cells and subjected them to an overnight cultivation in a culture medium. The objective behind this step was to foster a vast yield of the constructed plasmids through DH5α cloning.
Following that, we isolated the plasmids from the well-cultivated DH5α cells and subjected them to a second round of enzyme cuts, followed by gel electrophoresis to confirm the success of our plasmid construction. As depicted in figure 2, the gel electrophoresis results displayed two distinct bands at approximately 6000 base pairs and 760 base pairs. These bands corresponded respectively to the pET28a plasmid and the viral target segments.
With the success of our plasmid construction confirmed in the previous phase, we moved forward to express and purify the proteins. As DH5α lacks the ability to express proteins, we transferred the constructed plasmids into BL21 bacteria for protein expression. Following an overnight cultivation (as shown in figure 3), we proceeded to the next steps.
We selected specific bacterial colonies and initiated single clone cultivation. We added a molecule called IPTG to encourage these bacteria to produce the protein. To figure out the best conditions, we carried out some serious experimenting. We played around with two variables: temperature and the concentration of IPTG. Here's what our exploration looked like:
Unfortunately, there was a little hiccup, and we lost some data for the 37°C, 0.75mM group. Nevertheless, we moved on. After inducing the BL21 E. coli to express the protein, we went through ultrasonic disruption and centrifugation. Then, we ran a protein gel (figure 4) and used equipment to quantify our results.
Using the results from our modeling analysis, we decided to go with conditions involving 37°C and an IPTG concentration of 0.5mM for scaling up the culture. After expanding the cultivation, we once again went through the steps of ultrasonic disruption and centrifugation. Consulting the scientific literature, we learned that the COVID-19 RBD protein is inclusion body protein. In simple terms, it's like a protein cluster that forms inactive solid particles within the cells. This can be seen in figure 6 with the red circle.
Function Testing
Having obtained the purified protein, we proceeded to a crucial phase: the ELISA test. Our objective was to scrutinize the interaction between the RBD protein and the human ACE2 protein, a pivotal step in assessing the potential of our vaccine. This phase held the key to unveiling whether our project was on the right track.
In a meticulous sequence, we finished the ELISA test within a high-binding plate. Sequentially, we introduced the components – RBD protein, Biotinylated-ACE2, Streptavidin-HRP, and the TMB substrate solution. As this chemical symphony unfolded, a discernible change in solution color emerged, signifying the culmination of our experiment and its intrinsic success.
Moving forward, we quantified our success by measuring the OD450 values. By subtracting the values of the control group (0mg/ml), we accentuated the true essence of the experiment. The graphical representation of this processed data not only shows the trend but also clarifies the impact of the interaction.
Reference
- N. Zhu et al., A Novel Coronavirus from Patients with Pneumonia in China, 2019. N Engl J Med 382, 727-733 (2020).
- R. Lu et al., Genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding. Lancet 395, 565-574 (2020).
- P. Zhou et al., A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature 579, 270-273 (2020).
- A. C. Walls et al., Structure, Function, and Antigenicity of the SARS-CoV-2 Spike Glycoprotein. Cell 181, 281-292 e286 (2020).
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 499
- 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 499
- 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 784
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 499
- 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 499
Illegal AgeI site found at 268
Illegal AgeI site found at 670 - 1000COMPATIBLE WITH RFC[1000]
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