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.