Plasmid construction
The total RNA was extracted from the liver tissue samples of tilapia infected with TiLV as described in a previous study6 using GENEzol reagent (Geneaid Biotech, Taiwan). The RNA was reverse transcribed into complementary DNA (cDNA) using a cDNA synthesis kit (Toyobo, Japan). Specific primers targeting the TiLV S4 gene were designed based on the full-length sequence of TiLV S4 (accession number MK425013) for amplification by polymerase chain reaction (PCR). The TiLV S4 gene was amplified using the primers TVS4F 5’-ggatccatATGGTGAGAACTACAAAGAC-3’ and TVS4R 5’-gtcgactcgagCTATCTTCCAACAGCCCC-3’ by high-fidelity DNA polymerases (Thermo Fisher Scientific, USA). The specific PCR product was digested with NdeI/XhoI and then cloned into a linearized pET28a (+) expression vector, which yielded pTVS4 (non-optimized codon, NOC).
For the pTVS4 plasmid (codon-optimized, OC), the TiLV S4 nucleotide sequence was codon optimized, synthesized, and subsequently cloned into the expression plasmid pET28a (+) at the NcoI/XhoI sites by Synbio Technologies (USA). The codon adaption index (CAI) value was obtained from the self-developed NG Codon optimization software of Synbio Technologies (USA) available at the following link https://synbio-tech.com/codon-optimization-technology. The constructed recombinant plasmids were then transformed into an E. coli BL21(DE3) expression host and stored in aliquots with 40% glycerol at − 80 °C.
Expression and confirmation of the recombinant TiLV S4 protein
E. coli BL21(DE3) cells harboring pTVS4(OC) and pTVS4(NOC) were cultured at 37 °C overnight on Luria–Bertani (LB) agar plates (10 g/L tryptone, 10 g/L NaCl, 5 g/L yeast extract, and 15 g/L agar) supplemented with 50 µg/mL kanamycin. A single colony was then inoculated into 5 mL LB broth with 50 µg/mL kanamycin and incubated in a shaking incubator (200 rpm) at 37 °C overnight. Subsequently, 1% of the overnight culture was inoculated into 50 mL of a fresh LB medium with an appropriate antibiotic solution and incubated under the same conditions until the optical density at 600 nm (OD600) reached 0.4–0.6. One millimolar of isopropyl β-d-1-thiogalactopyranoside (IPTG) was added to induce protein expression, and the culture was further cultivated for 6 h at 37 °C. The OD600 values were measured to determine the relative growth of the postinduction culture.
The cell dry weight was calculated from the correlation between the OD600 and the actual cell dry weight, as shown in Supplementary Fig. S1. The cells were then collected by centrifugation at 12,000 × g for 10 min at 4 °C and resuspended in a lysis buffer at pH 7.8 with a ratio of 1:50 (g cell dry weight: mL lysis buffer). The cells were disrupted using sonication with a cycle of 5 s on and 10 s off for a total duration of 4 min. The lysate was subsequently collected by centrifugation at 12,000 × g for 10 min at 4 °C. The protein concentration in the supernatant was quantified using a bicinchoninic acid assay (Thermo Fisher Scientific, USA), and the recombinant TiLV S4 protein expression was analyzed using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The proteins were then transferred to a nitrocellulose membrane, which was subsequently blocked in a blocking buffer comprising 5% (w/v) skim milk in phosphate buffer saline (PBS) at 4 °C for 16 h. The membrane was then washed three times with PBS containing 0.1% (v/v) Tween 20 (PBS-T) and incubated with a 1:4000 dilution of horseradish peroxidase (HRP)-conjugated anti-His antibody (Bio-Rad, USA) in the blocking buffer for 1 h. Following incubation, the membrane was washed three times with PBS-T and incubated with an enhanced chemiluminescence (ECL) substrate. The developed protein bands were observed using the iBright™ CL1500 Imaging System (Invitrogen, USA).
Optimization conditions for recombinant TiLV S4 protein expression
E. coli BL21(DE3) containing pTVS4(OC) was cultured at 37 °C until it reached an OD600 value of 0.4–0.6. Then, the effects of the alternative inducers lactose and galactose on recombinant TiLV S4 protein expression were investigated at various final concentrations of 0.1, 0.55, and 1 mM. The bacterial cultures were subsequently incubated in a shaking incubator (200 rpm) at 37 °C for 6 h. The OD600 values were measured to determine the relative growth of the bacteria postinduction. Then, the biomass was harvested by centrifugation at 12,000 × g for 10 min at 4 °C. The expression of the recombinant TiLV S4 protein was examined using SDS-PAGE and Western blot analysis, as described in the previous section.
The effects of the induction conditions, that is, the temperatures (25 °C, 30 °C, and 37 °C) and times (0, 1, 2, 4, 6, 12, and 24 h postinduction) on recombinant TiLV S4 protein expression were investigated using the optimal inducer and induction concentration. The OD600 values were measured to determine the relative growth, and the expression of the recombinant TiLV S4 protein was evaluated using SDS-PAGE and Western blot analysis.
Optimization conditions for recombinant TiLV S4 protein purification
The recombinant TiLV S4 protein was expressed under optimal conditions. The bacteria cells were harvested by centrifugation at 12,000 × g at 4 °C for 10 min and then disrupted. After cell lysis, the supernatant containing the soluble TiLV S4 protein was collected and dialyzed overnight to transition from a lysis buffer to a binding buffer. Meanwhile, the cell pellet was washed three times with the binding buffer. It was resuspended in the binding buffer supplemented with 30 mM imidazole and a urea concentration of 6 M at a ratio of 1:50 (g cell dry weight: mL lysis buffer) and incubated at 4 °C for 16 h under denaturing conditions. Then, the samples were centrifuged at 12,000 × g for 20 min at 4 °C to precipitate the insoluble proteins and cell debris. Lastly, the supernatant containing the recombinant TiLV S4 protein was transferred to a new tube.
For purification, a 1:1 mixture of the soluble and denatured protein was loaded onto a 1 mL Ni-Sepharose 6 Fast Flow column (Cytiva, USA) and incubated on ice with gentle shaking for 1 h. Following incubation, the flow-through protein was collected, and the resin was washed with 2 mL binding buffer containing 3 M urea and 30 mM imidazole to remove the unbound proteins. The bound TiLV S4 proteins on the column were eluted in 0.5 mL fractions using stepwise increases in the imidazole concentrations of 50, 100, 200, 300, 400, and 500 mM. Afterward, each fraction eluted with imidazole was examined using SDS-PAGE and Western blot analysis, as previously described herein. The fractions containing the highest concentrations of TiLV S4 protein were pooled and subjected to dialysis (10,000 Da molecular weight cut-off) by serially passing them through binding buffers containing 2 M urea for 2 h and 1 M urea for 2 h before being incubated in binding buffer overnight.
Dot blot immunodetection assay
Two-fold serial dilutions of purified TiLV S4 protein, E. coli BL21(DE3) protein, and TiLV virus strain VETKU-TV0110 were prepared at concentrations of 100 ng/µL, and 2 µL of each dilution was dotted onto a nitrocellulose membrane. The membrane was left to dry for 15 min, blocked with a blocking buffer comprising 5% (w/v) skim milk in PBS for 1 h, and washed three times with PBS-T. The membrane was incubated with TiLV-infected fish serum and uninfected fish serum at a dilution of 1:100 in the blocking buffer for 1 h. After three washes with PBS-T, the membrane was incubated with a mouse anti-tilapia IgM antibody (Aquatic Diagnostics Ltd, UK) at a dilution of 1:1000 in the blocking buffer for 1 h. Following three washes with PBS-T, the membrane was probed with goat anti-mouse IgG HRP-conjugated antibody (SeraCare, USA) at a dilution of 1:2000 in the blocking buffer for 1 h and washed three times with PBS-T. After incubation with the ECL substrate, the band intensities were captured using the iBright™ CL1500 Imaging System.
The membranes were dotted with a serial dilution of purified TiLV S4 protein, bacteria protein, and TiLV virus and incubated with rabbit anti-TiLV IgG36 at a dilution of 1:1000 in blocking buffer for 1 h and mouse anti-rabbit IgG (Thermo Fisher Scientific, USA) at a dilution of 1:2000 in blocking buffer for 1 h. The wash, ECL substrate incubation, and image analysis were performed using the iBright™ CL1500 Imaging System.
Statistical analysis
The correlation of the relative growth of E. coli under various culture conditions was analyzed using the SPSS statistical program (version 26). Any statistical differences between the groups were compared using Duncan’s multiple range test with the significance level set at p < 0.05.
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- Source: https://www.nature.com/articles/s41598-024-83293-8