INTRODUCTION- A.hydrophila is a facultative
anaerobic, Gram-negative, and rod-shape bacterium that can live in a wide range
of temperatures, pHs, conductivities and turbidities, but favorably grows in
warm climates [1,2]. A.
hydrophila is considered as an opportunistic pathogen responsible for
numerous diseases in human such as gastroenteritis, skin
infections, peritonitis, bacteremia, hemolytic uremic syndrome, and necrotizing
fasciitis, etc [3,4]. Especially, this bacterium is also a
well-established fish pathogen that causes hemorrhagic
septicemia and red sore disease in carp, catfish, salmone, dogfish, and goby,
that results in huge global economic losses every year [1,5]. In
China, outbreaks of motile Aeromonas septicemia
were responsible for losses of 2,200 tons of dead fish per year [6].
In the summer of 2009, an outbreak of the same disease resulted in an estimated
loss of more than 12 million dollars in catfish farming in the southeastern
United States [7].
To prevent the infection of A.
hydrophila, commercial antibiotics are traditionally and popularly used in
fish farming [8]. However, the overuse of antibiotics has raised
concerns regarding the development of antibiotic resistance that seriously
affects public health [9,10]. Therefore, in previous studies, some
vaccines, including heat-killed bacteria [11], S-layer recombinant
protein [12], lipopolysaccharide [13], attenuated
bacteria [14], and biofilm oral vaccine [15]
were developed to control the spread of A.
hydrophila and also to
reduce the use of antibiotics. Recently, the bacterial outer membrane proteins
(Omps) have been gained attention as potential vaccines due to their high
immunoprotective capacities [16,17]. Importantly, among various Omps
in A. hydrophila, the vaccination with Omp38 can effectively protect fish
against this bacterium through the stimulation of both specific and
non-specific immune responses as demonstrated by Wang et al. [18].
For the
reasons mentioned above, in this study, we focused on establishing an E. coli strain that can express A. hydrophila Omp38 for the future
application in vaccine production. The recombinant Omp38 can also be used to
produce a specific antibody, which has not yet been available on market.
MATERIALS AND METHODS
Bacterial strains,
Medium and Other Reagents- The E. coli strains DH5a (F– F80lacZΔM15 Δ(lacZYA-argF)U169 recA1 endA1 hsdR17(rK–, mK+) phoA supE44 λ– thi-1 gyrA96 relA1) and BL21(DE3) (F– ompT hsdSB (rB–, mB–) gal dcm (DE3) (Thermo Fisher Scientific, USA) were used for the cloning and expression
of recombinant Omp38. The A. hydrophila strain
used in the current study for omp38 gene amplication was isolated from diseased fish in aquaculture of the Mekong Delta,
Vietnam. All bacterial strains were cultured at 37°C in LB broth (1% trypton,
0.5% yeast extract, and 0.5% NaCl) with reciprocal shaking (250 rpm). All
enzymes for DNA cloning were purchased from Invitrogen (USA). The DNA
ladder HyperLadderTM 1 kb and the Peptide Molecular
Weight Marker were purchased from Bioline (USA) and GE Healthcare (USA),
respectively. The other chemicals and reagents were obtained from Merck (USA).
Plasmid
construction- The coding
sequence of Omp38 (1008 bp) was amplified from A. hydrophila genome
using primer pair omp38-F (5’-GGA AGA TCT GGATCC GTC ATC TAT CAG GCC GAT GAC
GGC TCC AAC GTC G-3’) and omp38-R (5’-CCG CTC GAG CGG ATG GTG ATG GTG ATG GTG
GAA CTC GTA GCG CAG GCC GAG GTG GTA-3’). The plasmid pET-28a(+) (Novagen, USA)
was isolated from DH5a/pET-28a(+)
using EZ-10 Spin Column Plasmid DNA Miniprep Kit (Bio Basic Inc., Canada). The
PCR product was cloned into BamHI/XhoI sites of plasmid pET-28a(+) and in-frame
with polyhistidine tag (6xHis) sequence to construct pET-omp38 [19].
The ligation mixture was then transformed into DH5a cells and the transformed clones were screened
on 100 µg/ml ampicillin containing agar plates [20]. The recombinant
plasmids containing omp38 gene were selected using PCR and further verified by
Sanger sequencing.
Expression
of recombinant Omp38- The plasmid pET-omp38 was transformed into
BL21(DE3) cells to establish the strain BL21(DE3)/pET-omp38 that can express
the recombinant protein fused with 6xHis tag (6xHis-Omp38). A colony of
transformed cells was cultured in 100 ml LB medium containing 100 µg/ml
ampicillin at 37˚C with shaking (250 rpm) in an Erlenmeyer flask (300 ml).
When the culture reached OD ~ 0.8, 1 mM isopropyl b-D-1-thiogalactopyranoside (IPTG) was added to
induce the expression of 6xHis-Omp38. Cells were further cultured for 4 hours
and then 1 ml of the culture was centrifuged at 6,000 g for 10 minutes to
harvest cells. Cells were washing and re-suspended in 1 ml of 0.1 mM phosphate
buffer pH 7. After that, cells were disrupted by sonication and 500 µl of cell
lysate was centrifuged at 10,000 g for 5 minutes to separate the supernatant
and pellet fractions. The pellet fraction was then suspended into 500 µl of 0.1
mM phosphate buffer pH 7. Fifty microlitter of total cell lysate, supernatant
and pellet fractions were mixed with 10 µl sample buffer 6X (0.35 M Tris-HCl,
10% SDS, 36% glycerol, 0.6 M dithiothreitol, 0.012% bromophenol blue, pH 6.8) and heated at 100˚C for 10 minutes for
protein denaturation. After that, 10 µl of each sample was
applied to each lane of a polyacrylamide gel (12.5%) for SDS-PAGE analysis [21].
An His-probe antibody (H-3): sc-8036 (Santa Cruz Biotechnology Inc., USA) was
used to detect 6xHis-Omp38. We used Anti-mouse IgG (whole molecule)-Peroxidase
antibody (A-9044, Sigma-Aldrich, USA) as the second antibody, and the signal
was detected by ECLTM Prime Western Blotting Detection Reagent (GE
Healthcare AmershamTM, UK). The purity of Omp38 protein in pellet
fraction was determined using ImageJ software [22].
RESULTS
Amplification of omp38 gene and
construction of plasmid pET-omp38- For
the high expression of Omp38 protein, pET-28a(+) was chosen as a
vector to carry the target gene into E.
coli cells. The omp38 gene was inserted into BamHI/XhoI site of pET-28a(+) so
that this gene can be effectively expressed under the control of the very
strong T7 promoter.
With the purpose mentioned above, the omp38 gene was amplified from A.
hydrophila by PCR, with a BamHI and an XhoI at the 5’- and 3’-end,
respectively. Electrophoresis of PCR products showed that the length of
amplicons was approximately 1,000 bp that was in accordance with the
theoretical length of omp38 gene (Fig. 1A). The PCR products were then digested
with restriction enzymes BamHI and XhoI, yielding a DNA fragment with two
sticky-ends for DNA cloning.
The plasmid pET-28a(+) was also digested with the same restriction
enzymes, that generated a single DNA band between 5,000 and 6,000 bp in size
(Fig. 1B). After digested, PCR products and plasmids were ligated using T4 DNA
ligase and the ligation mixture was then transformed into DH5a
cells to select recombinant plasmids.
.jpg)
Fig.
1: Gene and plasmid preparation
(A)
ORF sequence of omp38 gene was amplified from A. hydrophila genome by PCR. Samples were as follows: 1- No
template control; 2- PCR products
(B)
Plasmid pET-28a(+)
Samples
were as follows: 1- Purified plasmid; 2- Plasmid digested with BamHI/ XhoI
The transformed cells were then spread on a LB agar plate
containing 100 µg/ml ampicillin. Ten colonies grown on this plate were randomly
chosen and subjected to colony-PCR with primers omp38-F/R (Fig. 2A). The electrophoresis result showed that DNA bands
representing omp38 gene were present
in 6/10 samples, suggesting that these colonies were successfully transformed
with pET-omp38.
We next isolated plasmids
from the above six omp38-positive clones and used them as templates for PCR
analysis with omp38-F primer and the T7-terminator primer (5’-GCT AGT TAT TGC
TCA GCG G-3’) to verify the insertion of omp38 gene into plasmid pET-28a(+). We found that all reactions from
these six plasmids produced a DNA band around 1,100 bp (Fig. 2B), that was
equal to the size of omp38 ORF plus the length from XhoI position to the
T7-terminator primer binding site. This result indicated that all these
plasmids contained omp38 gene with proper direction.
.jpg)
Fig.
2: Selection of recombinant plasmids
(A)
Obtained colonies were analyzed by PCR with primers omp38-F/R to select the
omp38-positive colonies. Samples were as follows: 1- No template control; 2-
PCR product from A. hydrophila genome
(positive control), 3 to 12- PCR products from ten randomly chosen colonies
(B)
Candidate plasmids were verified by PCR with omp38-F and T7 terminator primers.
Samples were as follows: 1- No template control; 2 to 7- PCR products from six
plasmids
DNA sequencing and
database search- The cloned omp38 gene was
sequenced using Sanger method and the result (Fig. 3) was submitted to a BLAST
server (http://www.ncbi.nlm.nih.gov/BLAST/) to check the sequence accuracy. The
interpreting BLAST output showed that there are some variety of Omp38 proteins
among A. hydrophila strains and the
omp38 sequence obtained in this study
was the same as those found in nine A.
hydrophila strains ZYAH72, GYK1, D4, JBN2301, NJ-35, J-1, pc104A, AL09-71,
and ML09-119 with 100% identity and 100% coverage. These data demonstrated that
we successfully and correctly inserted omp38 gene into pET-28a(+).
.jpg)
Fig.
3: DNA sequence of the omp38 gene
that was cloned in this study
Expression of Omp38
protein- The plasmid pET-omp38 was transformed
into BL21(DE3) cells to produce Omp38. The BL21(DE3) strain carries the gene
coding for T7 RNA polymerase under the control of lacUV5 promoter. The adding
of IPTG induces the expression of T7 RNA polymerase and subsequently stimulates
the expression of omp38 gene downstream of T7 promoter in pET-omp38 plasmid.
Therefore, a colony of transformed cells was cultured in LB medium
containing 100 µg/ml ampicillin and 1 mM IPTG was added to induce the
expression of recombinant protein. Cells were disrupted and then soluble
proteins and aggregates were separated and analyzed by SDS-PAGE method. Lysates
of BL21(DE3) cells and BL21(DE3)/pET-omp38 cells untreated with IPTG were used
as the negative controls. A clear protein band around 38 kDa appeared in
induced BL21 (DE3)/pET-omp38 sample and was absent in all negative controls
(Fig. 4A). Since omp38 protein is fused with 6xHis tag, this band could be
detected by western blot analysis with anti-Histidine antibody (Fig. 4B). These
results indicated that the addition of IPTG induced the expression of
6xHis-Omp38 protein and that we successfully established an E. coli strain that can produce Omp38
protein.
.jpg)
Fig.
4: The expression of 6xHis-Omp38 protein was analyzed by SDS-PAGE (A) and
Western Blot analysis (B)
Samples
were as follows: 1- Un-induced BL21(DE3); 2- IPTG-induced BL21(DE3); 3-
Un-induced BL21(DE3)/pET-28a(+); 4- IPTG-induced BL21(DE3)/pET-28a(+); 5-
Un-induced BL21(DE3)/pET-omp38; 6- IPTG-induced BL21(DE3)/pET-omp38, total
proteins; 7- IPTG-induced BL21(DE3)/pET-omp38, insoluble fraction, 8-
IPTG-induced BL21(DE3)/pET-omp38, soluble fraction
Additionally, we also found that this protein was mostly expressed
in inclusion body form since it was not observed in the soluble fraction of
cell lysate (Fig. 4A). The formation of such aggregates is considered to be
benefit for protein purification, as almost bacterial native proteins are
soluble. Accordingly, the ImageJ analysis of SDS-PAGE picture (Fig. 4A) showed
that the Omp38 protein comprised approximately 40% of total proteins in cell
lysate (well 6) and was present in insoluble fraction with high purity (~95%)
(well 7).
DISCUSSION- As stated in pET system manual (Novagen, USA), the pET plasmid
system is a very powerful tool for recombinant protein expression, which allows
the high expression level of target protein, up to 50% of the total cell
protein amount [19]. Therefore, in this study, we chose pET-28a(+)
plasmid for the expression of our target protein Omp38 and succeeded in
creating the E. coli strain BL21(DE3)
expressing 6xHis tag-fused Omp38 protein, which represented ~40% of total cell
proteins (Fig. 4A). This result was achieved under the current un-optimized
conditions in a 100 ml-flask scale and might be improved by the further
optimization and tighter control of medium, pH, oxigen concentration, and other
culture conditions using a fermenter.
In two previous studies, in order to evaluate the Omp38 protein as
a vaccine antigen against A. hydrophila,
Wang et al. [18]; Ni et al. [23] also used the
same system for Omp38 expression, and after purification using HisTrapTM
HP, the protein purities obtained were about 80% and 90%, respectively. We here
found that the target protein 6xHis-Omp38 was expressed mostly in inclusion
body form and comprised about 95% of the aggregated protein in E. coli cells (Fig. 4A, well 7).
Therefore, compared to the above studies, our target 6xHis-Omp38 protein can be
easily obtained with higher purity using a centrifuge. However, if a very high
purity is required (>95%), the recombinant Omp38 can be simply purified
using Ni-NTA affinity chromatography [24].
Additionally, Kasik et al.
[25] found that inclusion bodies carrying the E2
antigen of classical swine fever virus induced
both systemic and mucosal responses when being
fed to mice without any adjuvant. Therefore, it seems that the obtained Omp38
inclusion bodies can be directly used as an oral vaccine without any further
step. On the other hand, according to Ni et
al. [23] the omp38 gene
could be detected in 75% of the tested A.
hydrophila strains, suggesting that although Omp38 protein showed high
immunoprotective activity, the vaccination with Omp38 protein could protect
fish against most, but not all, A.
hydrophila strains. Therefore, an approach based on the combination of
Omp38 and some other outer membrane proteins should be considered to widen the
range of protected strains.
CONCLUSIONS- In this study, we successfully established an E. coli strain that could highly express Omp38 protein (making up
~40% of total proteins) when being induced by IPTG. The Omp38 protein was
expressed as inclusion bodies so that it could be easily obtained with high purity
(~95%) from cell lysate by centrifugation.
The inclusion body obtained in this study can be directly used as
a potential vaccine candidate for fish to control the spread of A. hydrophila or to produce a specific
antibody by immunization of animals. Since inclusion bodies are relatively
stable, the mixing of Omp38 aggregates and food for oral administration might
be an effective approach for labor-effective fish vaccination. Additionally,
the simple process to obtain high-purity Omp38, as shown in this study, might
also enable us to produce a cost-effective vaccine that can replace antibiotics
in the fish farming.
ACKNOWLEDGMENTS- This study was supported by a fund from University of Science,
Vietnam National University-Ho Chi Minh city, Vietnam.
CONTRIBUTION OF AUTHORS
Research design: Dang Thi Phuong Thao
Data collection: Le Thi Kim Phuong, Thi Hoa Rol, Nguyen Hieu Nghia
Data analysis and
Interpretation: Le Thi Kim Phuong, Nguyen
Thi My Trinh, Dang Thi Phuong Thao
Literature search: Le Thi Kim Phuong, Nguyen Thi My Trinh
Writing article: Nguyen Thi My Trinh
Critical review: Nguyen Thi My Trinh, Dang Thi Phuong Thao
Article editing: Nguyen Thi My Trinh
Final approval: Dang Thi Phuong Thao
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