Part:BBa_K4861008
pET-28a(+)-SARS-CoV-2-wuhan-RBD
The spike protein of the Wuhan variant 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. We used the restriction enzymes NcoI and XhoI to digest the SARS-CoV-2 RBD gene and the vector. Subsequently, the SARS-CoV-2 RBD fragment was ligated to the pET-28a vector backbone using T4 DNA ligase. Finally, the recombinant plasmid was transformed into Escherichia coli for screening.
Wuhan-pET28a BBa_K4861008
Construction Design
Wuhan-pET28a is a novel plasmid constructed using the pET-28a vector (BBa_K3521004 - parts.igem.org) and a gene fragment Wuhan-SARS-CoV2 RBD (BBa_K4861000 - parts.igem.org).
Engineering Principle
The S (spike) protein of neocoronaviruses is a key protein for their infection of the human body, which mediates viral entry into host cells by binding to the ACE2 receptor on the surface of the host cell, and the S protein is preferred for the design of recombinant protein vaccines against neocoronaviruses[1]. The S protein is divided into two subunits, S1 and S2, of which the S1 subunit contains a receptor-binding domain (RBD, receptor- binding domain), which is a key structure when the virus enters the host cell. Therefore, it is very appropriate to choose RBD as the target antigen for designing recombinant protein vaccines[2]. The code of Wuhan-SARS-CoV2 RBD represents a gene segment of the S protein from the Wuhan strain (COVID-19).
When this plasmid is introduced into BL21 Escherichia coli, it will produce the recombinant protein we need. It can be used to manufacture recombinant protein vaccines for viral strains.
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 overnight cultivation in a culture medium. The objective behind this step was to foster a vast yield of the constructed plasmids through DH5α cloning.
Figure 1. LB medium of DH5α for overnight culture
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.
Figure 2. Enzyme digestion validation of recombinant plasmids
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, we proceeded to the next steps.
Figure 3. LB medium for BL21 overnight culture
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 experiments. We played around with two variables: temperature and the concentration of IPTG. Here's what our exploration looked like:
Table 1. Optimization of IPTG induction conditions
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 and used equipment to quantify our results.
Figure 4. Protein gel results
Figure 5. Protein concentration of each group
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 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.
Figure 6. Inclusion body protein
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.
Figure 7. Changing color in solution on the high-binging plate
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.
Figure 8. Results of ELISA
Reference:
- [1] N. Zhu et al., A Novel Coronavirus from Patients with Pneumonia in China, 2019. N Engl J Med 382, 727-733 (2020).
- [2] R. Lu et al., Genomic characterization and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding. Lancet 395, 565-574 (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 BglII site found at 5195
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 NgoMIV site found at 3415
Illegal NgoMIV site found at 3575
Illegal NgoMIV site found at 5163
Illegal AgeI site found at 268
Illegal AgeI site found at 670 - 1000COMPATIBLE WITH RFC[1000]
None |