|Research Article (Open access)|
SSR Inst. Int. J. Life. Sci., 5(3): 2328-2334, May 2019
Cloning and Expression of Outer Membrane Protein Omp38 Derived from Aeromonas hydrophila in Escherichia coli
Le Thi Kim Phuong1, Nguyen Hieu Nghia2, Thi Hoa Rol3, Nguyen Thi My Trinh4, Dang Thi Phuong Thao5*
1PhD Scholar, Laboratory of Molecular Biotechnology, VNUHCM-University of Science, Vietnam
2Student, Laboratory of Molecular Biotechnology, VNUHCM-University of Science, Vietnam
3Student, Laboratory of Molecular Biotechnology, VNUHCM-University of Science, Vietnam
4Postdoctoral Researcher, Laboratory of Molecular Biotechnology, VNUHCM-University of Science, Vietnam
5Associate Professor, Laboratory of Molecular Biotechnology, VNUHCM-University of Science, Vietnam
*Address for Correspondence: Dr. Dang Thi Phuong Thao, Laboratory of Molecular Biotechnology, University of Science,Vietnam National University- Ho Chi Minh City, Ho Chi Minh city, Vietnam
ABSTRACT- Background: Aromonas hydrophila is an aquatic bacterium involved in various diseases in fish, resulting in serious economic losses every year. In previous studies, the outer membrane protein Omp38 was demonstrated to have high immunoprotection capacity, suggesting the use of this protein as a vaccine candidate to protect fish against A. hydrophila in fish aquacultures.
Methods: The gene coding for Omp38 was amplified from A. hydrophila genome and inserted into BamHI/XhoI sites of plasmid pET-28a(+). The recombinant plasmid was then introduced into E. coli BL21 (DE3). Transformed E. coli cells were treated with IPTG to induce the expression of Omp38 fused with 6xHis tag. The presence of 6xHis-Omp38 was detected by western blot analysis using an anti-Histidine antibody.
Results: We successfully constructed an E. coli strain that can highly express Omp38 protein (comprising ~40% of total cell proteins). The Omp38 protein was expressed as the form of inclusion bodies so that it could be easily obtained from cell lysate with high purity (³95%) by centrifugation.
Conclusions: This study was the first step in an attempt to produced Omp38-based vaccine to prevent the outbreak of diseases caused by A. hydrophila in fish framing.
Key Words: Aeromonas hydrophila, Escherichia coli, Omp38, Outer membrane protein, Recombinant protein
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 . 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 .
To prevent the infection of A. hydrophila, commercial antibiotics are traditionally and popularly used in fish farming . 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 , S-layer recombinant protein , lipopolysaccharide , attenuated bacteria , and biofilm oral vaccine  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. .
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 . The ligation mixture was then transformed into DH5a cells and the transformed clones were screened on 100 µg/ml ampicillin containing agar plates . 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 . 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 .
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.
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.
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(+).
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.
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 . 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. ; Ni et al.  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 .
Additionally, Kasik et al.  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.  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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