Gene synthesis of TgGRA8
The complete GRA8 coding sequence (accession number: TGME49_054720) was obtained from an online database (http://ToxoDB.org) . The TgGRA8 sequence consists of 810 nucleotides that encode a 269-amino acid protein. A signal peptide (SPs) of GRA8 was determined using online program, SignalP 4.1 (http://www.cbs.dtu.dk/services/SignalP/). The results showed that small fragments of amino acids 1-23 were expressed as a signal sequence. Therefore, encoding amino acid 24-269 was constructed and inserted into a pET-21a vector using NdeI and XhoI as the cloning sites (General Biosystems, USA).
Construction of recombinant TgGRA8
The potential transmembrane regions (TMs) of TgGRA8 were predicted by online server (http://www.cbs.dtu.dk/services/TMHMM/). The encoding amino acids 218-269 was transmembrane region. Therefore, only an antigenic fragment of recombinant TgGRA8 encoding amino acids 24–217 was PCR-amplified (Fig. 6). The primers used for amplification of the sequence by PCR was T7 promoter-(FW), 5′-TAA TACG ACT CAC TAT AG-3′ (New England Biolabs, UK); and TgGRA8-RW, 5′-AGT acc ggt GGT GGC GGT TGC CGG CTG-3′. The reverse primer was designed to contain the AgeI restriction site. PCR was performed using PCR Q5® High-Fidelity DNA Polymerase (New England Biolabs) using the following program: 98℃ for 1 min, followed by 30 cycles of 98℃ for 10 s, 58℃ for 20 s, and 72 ℃ for 20 s, and final extension at 72℃ for 2 min.
The PCR amplicon was digested using NdeI and AgeI. After digestion, the PCR product was ligated into the modified pET-21a vector harboring a C-terminal fusion FLAG tag (DYKDDDDK) (General Biosystems) and transformed into Escherichia coli DH5α-competent cells.
Ten colonies were selected and expanded in overnight cultures, and DNA was extracted using a QIAprep Spin Miniprep Kit (Qiagen, Germany). The insert of TgGRA8 in the purified plasmid was sequenced using a Dye Terminator Cycle Sequencing Kit (Applied Biosystems, USA) and the 3500xL genetic analyzer (Applied Biosystems). The TgGRA8 sequences were determined using Bioedit version 7.2.5 (Tom Hall Ibis Biosciences, USA).
Expression of TgGRA8
The recombinant TgGRA8 plasmids were transformed into E. coli strain Rosetta (DE3) cells and cultivated in 2XTY supplemented with 1% glucose and 200 ng/ml ampicillin at 37℃ with shaking at 200 rpm. E. coli carrying recombinant TgGRA8 was measured at an optimal density at 600 nm (OD600) of 0.5 and induced with isopropyl-β-D-thiogalactopyranoside at a final concentration of 1 mM 20 ℃ for various incubation periods (2, 4 and overnight) with shaking at 250 rpm. The induced bacteria were harvested via centrifugation at 4400 × g for 20 min at 4℃, and the bacterial pellet was resuspended in 20 ml of pre-chilled lysis buffer (150 mM NaCl, 50 mM Tris-HCL [pH 9.5], 1% Triton X-100, 1 mM EDTA [pH 8.0], and 1% NP 40) and then incubated at 4℃ for 30 min. After incubation, the bacterial cells were lysed via sonication on ice for 10 min, and 1 ml of 1× Protease Inhibitor Cocktail was added (Promega, USA). TgGRA8 expression was analyzed using 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
Anti-DYKDDDDK G1 affinity resin (GenScript, USA) was used for protein purification. The debris was centrifuged at 10,000 × g for 30 min at 4℃, after which the supernatant was transferred to a clean tube. The resin suspension (600 µl) was loaded into an empty gravity flow column (Bio-Rad, USA) and washed with Tris-buffered saline (50 mM Tris-HCl, 150 mM NaCl, pH 7.4). Protein was eluted from the resin using alkaline elution buffer (0.1 M Tris, 0.5 M NaCl, pH 12.0) and neutralized with 1 M HCl. The protein concentration was measured using NanoDrop ND-1000 UV/Vis spectrophotometer (Thermo Fisher Scientific, USA).
The eluted fractions were dialyzed using SnakeSkin Dialysis Tubing, 10 kDa cut-off (Thermo Fisher Scientific) against phosphate-buffered saline (PBS, pH 7.2) at 4℃. The debris formed during dialysis was removed via centrifugation at 10,000 × g for 5 min at 4℃, and the concentration of the purified recombinant protein was assayed using both SDS-PAGE and a Coomassie protein assay reagent kit using BSA according to the manufacturer’s protocol (Pierce Biotechnology, Inc., USA).
The immunoreactivity of the expressed TgGRA8 protein was determined by Western blotting using SDS-PAGE and then electrotransferred (Trans-blot, Bio-Rad) onto a nitrocellulose membrane (Millipore, USA). The membrane was washed three times with PBS, blocked with 5% skim milk, and then incubated at 37 ℃ for 1 h. After incubation, the membrane was washed three times with PBS containing 0.01% Tween 20 (PBS-T) and rinsed with PBS. The TgGRA8 protein in nitrocellulose membrane was probed using known reference positive and negative goat sera (diluted 1:250 in 5% skim milk) kept in our laboratory and incubated at 37℃ for 1 h. The membrane was washed three times with PBS-T and incubated with polyclonal mouse anti-goat immunoglobulin/HRP (Dako, Denmark) diluted 1:2000 in blocking buffer. The protein band was developed according to peroxidase activity using 3,3′,5,5′-tetramethylbenzidine (KPL, Gaithersburg, MD, USA).
Goat serum samples
The process of sample collection was reviewed and approved by the Animal Care and Use Committee of the Faculty of Veterinary Science, Mahidol University, Thailand (Approval No. MUVS-2018-03-09). A total of 306 serum samples were obtained from a goat farm in Kanchanaburi province, Thailand. The goats were restrained by holding the base of the horn and blood was collected from the jugular vein and immediately transferred into 10 ml vacuum blood tubes without anticoagulant. The animals were not allowed returned to their cage until complete hemostasis has been achieved. All blood samples were kept in cooled box with ice pack and sent to the laboratory at Faculty of Veterinary Science, Mahidol University. The sera were separated after sedimentation of blood cells and stored at −20 °C until examination.
Purified recombinant TgGRA8 was diluted at a final concentration of 0.1 µg/ml in coating buffer (50 mM bicarbonate, pH 9.6) and added to separate wells of the ELISA plates (Nunc, Denmark). The coated plates were incubated overnight at 4℃. The next day, the plates were washed five times with PBS-T and blocked with 5% PBS-skimmed milk (PBS-SM) for 1 h at 37°C. After washing with PBS-T, duplicate serum samples were diluted 1:250 in PBS-SM, and 50 µl of diluted serum were added to each well. The plates were incubated at 37 °C for 1 h and washed with PBS-T five times. Specific IgG antibody was detected using horseradish-peroxidase-conjugated anti-goat IgG antibodies (Invitrogen, USA). The conjugate was diluted 1:5000 with PBS, and 50 µl of diluted conjugates were added. After incubation at 37 °C for 1 h, the plates were washed five times with PBS-T, and then 3,3′,5,5′-tetramethylbenzidine (Invitrogen, USA) was added to develop the color. After 15 min, the reaction was stopped by adding 50 µl of 0.1 M HCl. OD450 was read using a microplate reader (model ELx808, Biotex, VT, USA).
The negative and positive control sera were confirmed using MAST® TOXOREAGENT (Mast Group, Liverpool, UK). Positive samples were considered when agglutination was observed at a dilution of 1:32 or greater.
The results of iELISA and LAT were calculated using online software (http://vassarstats.net) to determine the percentage of agreement, sensitivity, specificity, and the kappa values with 95% confidence intervals. The strength of agreement was graded as fair (κ = 0.21–0.40), moderate (κ = 0.41–0.60), and substantial (κ =0.61–0.80).