Results

Overview

Our primary aim is to design a recombinant protein vaccine. In the last few days, we obtained four recombinant proteins targeting the four types of COVID-19 virus: Wuhan, Delta, BQ1.1, and xBB1.5. Our experiment could be separated into three parts:

1. Plasmids construction

2. Protein expression and purification

3. ELISA test

 

 

We inserted the target fragment into the plasmid vector pET28a through enzyme cleavage and ligation. By cultivating in large quantities within E. coli BL21, we obtained a bacterial culture containing the target protein. Finally, the target protein was extracted and purified using a nickel column, which gives us the pure target proteins. In order to test the validity of the proteins, we test it with ELISA.

Experiment Results

Step 1: Plasmid Construction

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 four viral strains – Wuhan, Delta, BQ.1.1, and XBB.1.5 – 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. The cultured DH5 α  are shown in Figure 1

 

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 .1 , 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. This visual confirmation signifies the achievement of our plasmid construction endeavor and sets the stage for the subsequent protein expression phase.

 

Figure 2 .1  gel electrophoresis results

Enzyme digestion validation showed that we have got the correct recombinant plasmid. Subsequently, for further confirmation, we sent the recombinant plasmid to a biological company (Azenta) for sequencing, and the sequencing results are shown in the figure 2.2. This visual confirmation signifies the achievement of our plasmid construction endeavor and sets the stage for the subsequent protein expression phase.

 

Figure 2 . 2 . Sequencing results

Step 2: Protein Expression and Purification

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.

 

Figure 3. LB medium for BL21 overnight culture

Inducing Protein Expression

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 rigorous experiment s . We played around with two variables: temperature and the concentration of IPTG. Here's what our exploration looked like:

 

U nfortunately, 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 (figure 5).

 

F igure 4. SDS-PAGE gel results

 

F igure 5. Protein concentration of each group

Extended culture

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 an 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.

 

F igure 6. inclusion body protein

Renewing the Protein's Structure

Since our protein was in inclusion bodies, we went the extra mile for refolding. We used something called dialysis bags to gradually lower the concentration of an external solution. This helped remove the denaturing agents and allowed the inclusion bodies to regain their natural structure.

Purification with a Nickel Column

For the next step, we went for purification using a nickel column. Due to a special "His" tag on the pET28a plasmid, our protein had a soft spot for nickel. After a couple of thorough washes, we eluted the purified RBD protein from the nickel column.

Final Measurements

Wrapping things up, we measured the concentration using A280 readings. Here's what we found for each strain – Wuhan, Delta, BQ1.1, and XBB1.5: 0.001mg/ml, 0.007mg/ml, 0.012mg/ml, and 0.001mg/ml respectively. These measurements set the stage for calculating the dilution gradient for ELISA testing.

 

ELISA test

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 in figure 8 not only shows the trend but also clarifies the impact of the interaction.

 

Figure 8 . Curves of the OD450 values of each group against the concentration of RBD

 

A Definitive Achievement

To encapsulate our findings, our endeavors bore a definitive revelation. We not only successfully produced the RBD protein but also conclusively demonstrated its binding affinity with the human ACE2 protein across the Wuhan, Delta, BQ1.1, and XBB1.5 strains. This discovery holds profound implications, illuminating a potential avenue towards the development of a vaccine.

 

Future Plan

While we acknowledge that our current capabilities and time constraints may have introduced a degree of experimental error, we aspire to advance our research in the future with a sharper focus on precision. We aim to mitigate experimental inaccuracies by employing stricter protocols and more sophisticated laboratory equipment. This pursuit is driven by our commitment to enhancing the quality of experimental results, increasing protein yield, and elevating purity levels.

 

Furthermore, we are acutely aware that vaccine development is an arduous and protracted process, and our current work represents just a fraction of this extensive journey. In the subsequent phases of our research, we intend to extend our efforts towards manufacturing subunit vaccines, gradually progressing to safety testing in animals. Ultimately, our goal is to develop a recombinant protein vaccine for COVID-19, bolstering humanity's defenses against this virus.

 

Lastly, our experiments have illuminated the advantages of recombinant protein vaccines. These vaccines boast formidable safety profiles, cost-effectiveness, and the capacity to induce humoral immunity while also activating cellular immunity. As a result, we envision that recombinant protein vaccines may emerge as a versatile defense against a spectrum of infectious diseases in the future.

 

In summary, our current work represents a stepping stone towards a broader and more impactful journey. With a commitment to precision, a clear vision of the future, and an appreciation for the versatility of recombinant protein vaccines, we remain steadfast in our pursuit of safer and more effective defenses against infectious diseases. Our iGEM journey is a reflection of our shared commitment to discovery and growth.