Overview

The aim of the experiment is to produce a Recombinant Protein Vaccine with Spike-Protein as an antigen in E. coli and assesse its functionality. To achieve this idea, we design the following methods:

 

Each of these components came with its own set of challenges and obstacles, both in the design process and during practical implementation.

 

Engineering Cycle

1.  Design

Spike protein is the key protein for Covid-19 to infect the human body. It mediates the entry of the virus into host cells by binding to the ACE2 receptor on the surface of host cells.(R. Lu et al, 2020) Therefore, spike protein is the preferred choice for designing recombinant protein vaccine against Covid-19. The spike protein is divided into two sub-units (P. Zhou et al., 2020), S1 and S2, in which the S1 sub-unit contains a receptor binding domain (RBD), which is the key structure for the virus to enter the host cell.(A. C. Walls et al., 2020) Therefore, it is appropriate to select RBD as the target antigen for designing recombinant protein vaccines.

The Recombinant Protein Vaccine was planned to combine the Spike protein in E. coli with the four RBD fragments of the SARS-CoV-2 virus(Wuhan, Delta, BQ1.1, XBB1.5). Using RBD as an immunogen produces Anti-RBD antibodies after vaccination and finally inhibits virus invasion and infection(Figure 1.1).

 

 

Figure 1.1 Diagram of Spike Protein combines hACE2 through RBD and produce anti-body

 

The experiment will be separate d  into three parts.

In the first part, we will build sample plasmids and prove they are  successfully built. We used  the pET-28a(+) vector as the viral vector and transferred the RBD fragments in the four virus samples into the pET-28a(+) vector by enzyme digestion and enzyme chaining. RBD samples will go through Gel electrophoresis of nucleic acids to assess their success.

In the second part, we will transfer the plasmid samples to BL21(DE3) Competent E. coli. We will induce plasmid with the Isopropyl β-D-1-thiogalactopyranoside induction in different concentrations and temperatures. To test the optimal concentration for the plasmid, we will use SDS-PAGE discontinuous electrophoretic system and record the optimal concentration. We will purify the sample with His-Tag.

In the third part, we use the ELISA Test to assess the success of Anti-RBD antibodies.

 

2.  Build

Part I -Plasmid Construction

In building experiment, the pET28a(+) vector was employed as the expression vector throughout the experimental procedures (Figure 2.1). Initially, four viral cultures were cultivated for 12-16 hours in LB medium, and subsequently, the Vazyme DC201 Protocol was employed to extract the respective plasmids. Evaluation of the plasmid concentrations facilitated the selection of the most suitable plasmid for subsequent double digestion, targeting a specific region within the pET28a(+) vector (Figure 2.2). The insertion event was targeted at approximately 6000 base pairs of the pET28a(+) vector.

 

 

Figure 2.1 Diagram of pET28a(+) vector

 

 

Figure 2.2 Diagram of the process of double digestion

 

 

Part II -Cells Transformation

Two distinct strains of Escherichia coli were utilized in this study: DH5α and BL21. DH5α was specifically chosen for cloning purposes. Initially, the plasmid was transferred into DH5α cells. Following that  the plasmid was subsequently transferred into BL21 cells to facilitate protein expression (Figure 2.3).

 

Figure 2.3 Diagram of the process of pET28a(+) plasmid transform in BL21(DE3)

 

3.  Test

In order to prove that every step of the experiment is successful, we need to prove the success of each step through different verification methods.

 

Test I- Plasmid Construction Validation

In order to prove that four recombinant plasmids ( Wuhan-pET28a , Delta-pET28a, BQ1.1-pET28a, XBB1.5-pET28a) were digested successfully. According to the electrophoretogram(figure 3.1) , t he criterion for judging success is two parallel lines at 760bp and 6000bp.

 

 

Figure 3.1  E lectrophoretogram consist of highlight marker(M), Wuhan-pET28a  plasmid, Delta-pET28a plasmid, BQ1.1-pET28a plasmid, XBB1.5-pET28a 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  3 .2. This visual confirmation signifies the achievement of our plasmid construction endeavor and sets the stage for the subsequent protein expression phase.

 

Figure 3 .2. Sequencing results

 

Test II- Protein Expression and   O ptimal E xpression C oncentration of Determination

 

Protein expression was induced with the use of IPTG reagent, and an analysis of IPTG data allowed for the determination of the optimal concentration for protein expression. To this end, seven different concentrations of IPTG (0.002 mol/L, 0.001 mol/L, 0.00075 mol/L, 0.0005 mol/L, 0.00025 mol/L, 0.0001 mol/L) were introduced into 100 ml culture medium, with overnight cultures performed at 16 ° C and 3-hour cultures at 37 ° C (Figure 3.3 ). Subsequently, based on the conditions yielding calculates the optimal IPTG concentrations.

 

Figure 3.3  Diagram of the process cell expression

 

Based on the SDS-PAGE results   (figure 3. 4 ) through E-Gel Imager and the relevant A280 data(figure 3. 5 & 3.6 ) , we could conclude that the optimal concentration of IPTG to induce protein expression in this experiement is 0.5 mM.

 

Figure 3. 4   E lectrophoretogram of IPTG induction in 16°C and 37°C condition

 

 

 

Figure 3. 5  Chart of protein concentration in 16°C condition

 

Figure 3. 6  Chart of protein concentration in 37°C condition

 

 

Test III-ELISA Essay

The Enzyme-Linked Immunosorbent Assay (ELISA) is employed for testing and assessing the binding affinity between the SARS-CoV-2 RBD and the human ACE2 protein.

We immobilize the RBD onto an ELISA high binding plate, resulting in the generation of S2 Biotin. Subsequently, S2 Biotin binds to Streptavidin-labeled HRP (horseradish peroxidase) conjugate. Upon the addition of Tetramethylbenzidine (TMB) substrate solution, a colorimetric reaction occurs. We then measure the Optical Density (OD) and record the data (Figure 3. 7 ). This process allows us to quantify the interaction between these proteins with precision and biological relevance.

 

Figure 3. 7  The graph records the product reaction in 450nm OD

 

4.  Learn

Improvement I

Plasmid c onstruction and e xpression p rocess p roceeded s moothly, but f urther c odon o ptimization or t ranscription- t ranslation o ptimization of the p lasmid could be studied and  i mplemented to e nhance p rotein e xpression from the g ene s equence.

Improvement II

While r elative o ptimal e xpression c onditions were i dentified under the g iven s ettings, the u ltimate c onditions for p rotein e xpression may r equire f urther e xploration to specify more details, e specially c onsidering the u pcoming l arge- s cale i ndustrial p roduction, where c ost- e fficiency is c rucial. Further r esearch is n eeded to a chieve s ufficient p rotein y ield under c ost- e ffective c onditions.

Improvement III

According to the ELISA curves, we could see the trend that clarifies the certain impact of the interaction between these 4 groups of  SARS-CoV-2 RBD and the human ACE2 protein.  However, the e fficacy of SARS-CoV-2 RBD p roduced in Prokaryotic Cells to i nduce n eutralizing a ntibodies r equires f urther v alidation and e xperimentation. Further e xperiments are r equired to d etermine the a bility of the p roduced RBD to e ffectively i nduce n eutralizing a ntibodies.