Materials
AHPND strain of Vibrio parahaemolyticus XN89 and non-AHPND strain XN8 were kindly provided by Dr. Saengchan Senapin National Center for Genetic Engineering and Biotechnology (BIOTEC), Thailand. Vibrio cholerae, Vibrio vulnificus, Vibrio alginolyticus, and White Spot Syndrome virus were kindly provided by Dr. Tuan V. Vo from University of Agriculture and Forestry, Ho Chi Minh City, Vietnam. Female New Zealand white rabbits were provided by Central Animal House of Pasteur Institute in Ho Chi Minh City. PCR kit was product of Bioline, USA. Restriction endonucleases, DNA, and protein markers were all purchased from Thermo Scientific, USA. Ni-sepharose and Hitrap protein G HP column were purchased from Cytiva Life Sciences, Sweden. Culture media were purchased from BD. All other used chemicals were of analytical grade, unless otherwise specified.
Bacterial strains and growth conditions
All Vibrio strains were grown routinely in tryptic soy broth (BD Difco) containing 1.5% NaCl at 30 °C.
Preparation of recombinant PirBvp protein
Amplification and cloning of PirBvp
Plasmid DNA extracted from V. parahaemolyticus XN89 by using commercial plasmid isolation kit. Due to NdeI restriction site located within PirBvp gene, recombinase-free cloning in which designed primers with 15-bp overlap homology at their ends to cloning vector was exploited. The designed primers used for the amplification of PirBvp gene included ToxB-F (5’-taagaaggagatataCATATGACTAACGAATACGTTGTAAC-3’) and ToxB-R (5’-gtggtggtggtggtgCTCGAGCTTTTCTGTACCAAATTCATC-3’). The resulting amplicon was incubated with NdeI-XhoI-treated pET22b vector at RT for 30 min, then transformed into chemically competent Escherichia coli DH5α cells. The recombinant plasmid was confirmed for accurate insertion by both restriction enzyme digestion and sequencing.
Expression of recombinant PirBvp protein
The pET22b-PirBvp plasmid was transformed into E. coli BL21 (DE3) [10]. Colonies of E. coli BL21 (DE3) containing the pET22b-PirBvp plasmid was inoculated into 10-mL Luria broth media containing 50 μg/mL ampicillin and incubated overnight at 37 °C with shaking at 200 rpm. The expression of recombinant protein (rPirBvp) was induced by 0.5 mM IPTG and harvested by centrifugation. A volume of 1.5 mL bacterial broth was pelleted, mixed with 300 μL PBS, sonicated, and centrifuged to collect total protein, soluble protein, and insoluble protein fractions. Expression of the recombinant protein was confirmed by SDS-PAGE with Coomassie blue staining and Western blotting probed with anti-6xHis tag. Control samples were total fraction of E. coli BL21 (DE3)/pET22b with IPTG induction and E. coli BL21(DE3)/pET22b-PirBvp non-IPTG induction. After that, E. coli BL21 (DE3)/pET22b-PirBvp strain was expressed and scaled up to 100 mL LB medium for collecting rPirBvp. Soluble protein fraction was purified by affinity chromatography method with Histrap HP column (Cytiva Life Sciences). The column was loaded with 20 mL total soluble fraction. Next, the column was washed with binding buffer (20 mM phosphate 0.5M NaCl, 40 mM imidazole, pH 7.4). Finally, the target protein was eluted with elution buffer (20 mM phosphate, 0.5M NaCl, 108 mM imidazole, pH 7.4). The result of purification was verified by SDS-PAGE with silver staining and analyzed by a gel analyzer software. The concentration of obtained protein was determined by the Bradford method.
Immunization of rabbits and production of polyclonal antibodies
Healthy, 12-week-old, female New Zealand rabbits were maintained in the experimental animal facility, and experiments were performed in accordance with the Directive 2010/63/EU guideline approved by The Animal Care and Use Committee of University of Science, VNU-HCM in Ho Chi Minh City (ethical code 12/18-0599-01). The animals were housed singly in suspended cages and fed chow and water ad libitum. Three rabbits were injected subcutaneously with an emulsion of 1 mL rPirBvp in PBS, and 1 mL of Complete Freund’s Adjuvant at the first dose. Four booster injections of the same protein mixed with Incomplete Freund’s Adjuvant were given to each rabbit on a monthly basis. The rabbits were bled via the marginal ear vein prior to the first dose and two-week intervals, and serum was tested to detect antibodies against rPirBvp. Rabbits after completion of the experiment were anesthetized subcutaneously with Xylazine (5 mg/kg) for 5 min then Ketamine (40 mg/kg) followed by exsanguination to euthanize. After experiments, the rabbits were disposed and cremated following local regulations.
Detection of antibody by the Ouchterlony double immunodiffusion technique
Antibodies were detected using double immunodiffusion [11]. Petri plates containing casted agarose with six peripheral wells contained serially diluted serum, and a central well contained recombinant and native PirBvp antigen. Antigen and serum were filled into wells (20 μL per well). The double diffusion plates were stored in a humidified chamber at 4 °C, and the precipitin lines were examined daily for 2 days.
Detection of antibody by ELISA
Immunized rabbit sera after four injections were detected by coating the recombinant and native PirBvp proteins at 0.5 μg/mL in 0.1 M carbonate buffer, pH 9.6 overnight at 4 °C into 96-well ELISA plates. The plates were washed four times with PBS-0.01% Tween 20 (PBS-T) and blocked with 1% bovine serum albumin in PBS for 30 min at 37 °C. After a washing step, 100 μL of 1:1000 diluted serum was added to triplicate wells, and the wells were incubated for 1 h at 37 °C. Unbound antibody was removed by washing four times with PBS-T, and 100 μL of 1:40,000 diluted goat anti-rabbit IgG peroxidase conjugate (Sigma-Aldrich) was added to each well and incubated for 30 min at 37 °C. After washing four times with PBS-T, the wells were reacted with 3,3′,5,5′-Tetramemethylbenzidine (TMB) at room temperature in dark room for 10 min. The reaction was stopped by 50 μL 1N H2SO4. Absorbance was measured at 450 nm with a microplate reader.
Purification and determination of affinity-purified antibodies
Immunized rabbit sera were collected and precipitated by 50% ammonium sulphate. After dialysis against PBS, immunoglobulins were purified by Hitrap protein G HP column (Cytiva Life Sciences). Immunoglobulins were applied through affinity column. Next, the column was washed with binding buffer until the absorbance reached the baseline. Then, the respective bound antibodies were then eluted with 0.1 M glycine (pH 3.0) into 0.1 M Tris (pH 11.0), thus minimizing exposure of the antibody to acid. The effluent was dialyzed against PBS, determined content by Bradford assay and analysed by SDS-PAGE. Finally, affinity-purified antibody titer was determined by the ELISA method as described above. The titer of each antibody sample was arbitrarily designated as the maximum dilution that yielded at least twice the absorbance of the same dilution of nonimmune control antibody [12].