O
verview
Norovirus is characterized by high infectivity, diverse modes of transmission,
and easy mutation of the pathogen, and is the main pathogen of sporadic and fulminant acute gastroenteritis
in the population. It causes a huge disease burden to society, and there is no vaccine for norovirus in the
market at present. Therefore, our team is attempting to produce a vaccine against norovirus and bivalent
vaccines against norovirus and rotavirus at the same time.
To
achieve this goal, we first
genetically engineered the major antigenic genes of norovirus or (and) rotavirus into vector plasmids and
transformed them into
E. coli
BL21(DE3) or
Nissle 1917
, and then induced the plasmids to express the proteins upon culture. Finally,
we purified and analyzed the proteins.
Thus, we have provided some new parts and added new functional data to
existing parts, which are shown in the table below:
|
Part Contributions
|
||
|
Part Number
|
Part Name
|
Contribution Type
|
|
GST
|
New experimental data to an existing Part
|
|
|
RV VP7
|
New experimental data to an existing Part
|
|
|
GII.4-VP1
|
New basic part
|
|
|
GII.17-VP1
|
New basic part
|
|
|
RV VP7-GII.17-VP1
|
New composite part
|
|
|
pGEX-GII.4-VP1
|
New composite part
|
|
|
pGEX-GII.17-VP1
|
New composite part
|
|
|
pGEX-RV VP7-GII.4-VP1
|
New composite part
|
|
|
pGEX-RV VP7-GII.17-VP1
|
New composite part
|
|
1.
Add new
experimental
data to an existing part:
GST
BBa_K608408
In the construction of plasmids containing the norovirus and rotavirus antigen
genes, we fused and expressed a GST tag (
BBa_K608408
) at the C-terminus of the genes, which was designed to increase the soluble
expression of the proteins and to allow for subsequent facile purification of the target proteins. At the
same time, we also inoculated strains without the target gene,
i.e.
, containing only the GST tag, and after IPTG-induced expression, we
ultrasonically crushed the bacterium to obtain a protein lysate and purified it. SDS-PAGE results showed
that we successfully expressed and purified the GST protein (25 kDa).
Figure
1
SDS-PAGE results of GST
protein
expression
and purification
2.
Add new functional data to an existing part:
RV-VP7
BBa_K3992000
and
Create new parts
BBa_K4872009
and
BBa_K4872013
In order to
develop specific vaccines against norovirus
and
rotavirus
viruses
both, we introduced RV-VP7 (
BBa_K3992000
) to play a role in rotavirus vaccine function and then we constructed
RV
-
VP7-GII.17-VP1 (
BBa_K4872009
)
and plasmid
pGEX-RV-GII.17-VP1
(
BBa_K4872013
)
. RV structural protein vp7, on the outermost layer of virus particles, is the
first choice for the development of genetic engineering vaccines.
As shown in Figure 2, we successfully introduced the RV-VP7 fragment (898 bp)
into the plasmid.
Figure
2 PCR amplification result and sequencing result of plasmid construction:
pGEX-RV-GII.17-VP1
-
BBa_K4872009
and
BBa_K4872013
To obtain a bivalent vaccine against norovirus and rotavirus, we used a linker
that connects the GII.17-VP1
(
BBa_K4872002
)
antigenic gene of norovirus
to RV VP7 of rotavirus, and thus, we obtain
RV
-
VP7-GII.17-VP1 (
BBa_K4872009
)
.
To construct plasmid
pGEX-RV-GII.17-VP1
(
BBa_K4872013
)
, w
e
first
amplified
the antigen gene
RV VP7 and GII.17-VP1 us
ing
the PCR amplifier (
Figure
3
A
). Then,
the
RV VP7
and GII.17-VP1 fragments
underwent homologous recombination with the pGEX-4T-1 plasmid vector to obtain the recombinant plasmid
pGEX-RV-GII.17-VP1
(Figure
3
B)
. As shown in Figure
3
C-D, the colony PCR and sequencing results confirmed the successful
construction of the plasmid.
Figure
3
Construction
of plasmid pGEX-RV-GII.17-VP1
We inoculated the transformant, induced its expression, and explored its
optimal expression conditions.
To find the optimal conditions for the highest protein expression, we chose
different concentrations (OD
600
=0.3/0.6/0.8/1) of the bacterial solution and different IPTG induction times
(0 h/4 h/8 h/16 h). After obtaining each protein lysate, we measured the total protein amount (A280) under
different induction conditions (
Figure
4
A
). In addition, we examined the expression of the target proteins
(119 KDa)
using SDS-PAGE (
Figure
5
) and used the ImageJ software to quantify the target bands on the SDS-PAGE
gel, collected and organized the data, and plotted a line graph with OD
600
as the x-axis and gray value
as the y-axis (Figure
4
B).
As shown in Figure
4
, the protein concentration roughly tended to increase with increasing
bacterial concentration at the same IPTG induction time. When the bacterial concentration was 0.8 and the
induction time was 8 hours, the best protein expression level was achieved, indicating that this condition
was more suitable for expressing more target proteins.
Figure
4
Effect of
IPTG induction time and bacterial concentration on protein concentration
Figure
5
SDS-PAGE
results of RV-GII.17-VP1 protein under different expression conditions
Finally,
we
transformed
plasmid
pGEX-
RV-
GII.17-VP1
into
E. coli
Nissle 1917. Through the SDS-PAGE gel map, it was found that we have weak
bands within the
119
kDa
range, which means that RV-
GII.17-VP1
protein expression level is
weak in
E. coli
Nissle 1917
(Figure
6
)
.
This initially confirmed that
RV-GII.17-VP1 could be successfully expressed in Nissle 1917, but the expression conditions need to be
optimized.
Figure
6
SDS-PAGE
results of pGEX-RV-GII.17-VP1 protein expression in
E. coli
Nissle 1917
3.
Create new parts
BBa_K4872001
and
BBa_K4872010
GII.4-VP1 gene (
BBa_K4872001
)
is
from VP1 genes
of
the n
orovirus
mutant strain
.
To construct the
pGEX-GII.4-VP1
(
BBa_K4872010
),
we
amplified
the antigen gene GII.4-VP1 us
ing
the PCR amplifier
(
Figure
7
A
). Then,
the GII.4-VP1 fragment and the pGEX-4T-1 plasmid vector were digested with
restriction endonucleases
Eco
RI and
Xho
I, followed by the ligation using T4 DNA ligase to obtain the recombinant
plasmid pGEX-GII.4-VP1
(Figure
7
B)
. As shown in Figure
7
C-D, the colony PCR and sequencing results confirmed the successful
construction of the plasmid.
Figure
7
Construction of plasmid
pGEX-GII.4-VP1
We inoculated the positive transformant and induced protein expression by
IPTG. After obtaining protein lysate, we purified the target protein using a GST tag and verified the
protein expression and purification results by SDS-PAGE. The results are shown in Figure
8
, we successfully expressed GII.4-VP1 protein
(60 KDa)
, but the purified protein was barely visible, which might be due to the low
expression of protein.
Figure
8
SDS-PAGE results of pGEX-GII.4-VP1
protein expression
(P represents
" Precipitation" ,
S represents
" Supernatant" ,
T represents
" Flow through" ,
W represents
" Wash solution" and
E represents
" Eluent
"
)
3
.
Create new parts
BBa_K4872002
and
BBa_K4872011
GII.17-VP1 gene (
BBa_K4872002
)
is
from VP1 genes
of
the n
orovirus
mutant strain
.
To construct the
pGEX-GII.
17
-VP1
(
BBa_K4872011
), w
e
first
amplified
the antigen gene GII.17-VP1
us
ing
the PCR amplifier (
Figure
9
A
). Then,
the GII.17-VP1 fragment and the pGEX-4T-1 plasmid vector were digested with
restriction endonucleases
Eco
RI and
Xho
I, followed by the ligation using T4 DNA ligase to obtain the recombinant
plasmid pGEX-GII.17-VP1
(Figure
9
B)
. As shown in Figure
9
C-D, the colony PCR and sequencing results confirmed the successful
construction of the plasmid.
Figure
9
Construction of plasmid
pGEX-GII.17-VP1
We inoculated the positive transformant and induced protein expression by
IPTG. After obtaining protein lysate, we purified the target protein using GST tags and verified the protein
expression and purification results by SDS-PAGE. As shown in Figure
10
, we successfully expressed and purified GII.17-VP1 protein (60 kDa).
Figure
10
SDS-PAGE results of pGEX-GII.
17
-VP1
protein expression
(Prepresents
" Precipitation" ,
Srepresents
" Supernatant" ,
Trepresents
" Flow through" ,
Wrepresents
" Wash solution" and
Erepresents
" Elutent
"
)
