Vector construction
ELP fusion expression vector pET-ELP was constructed by cloning ELP coding sequence into pET-30a (+) vector (Novagen, USA) with NdeI and SacI digestion [15]. The coding sequences for the Cap protein of PCV2b (GenBank accession: GQ359004) and the VN epitopes [16] of PCV2a (GenBank accession: GQ359003), PCV2d (GenBank accession: GU001710) and PCV2e (GenBank accession: GU001709) were adapted to E. coli codon usage using JAVA Codon Adaption Tool [17]. The synthetic sequence, with a tobacco etch virus (TEV) protease recognition signal introduced at the 5′ end, was cloned into pET-ELP vector with HindIII and XhoI digestion. For the comparison purpose, the synthetic sequence was amplified by PCR using the forward primer (5′-CAGTACATCAAAGCTAACTC-3′) and the reverse primer (5′-CGGGTTCAGCGGCGGGTCTTT-3′), and cloned into pET-30a vector with NdeI and SalI digestion. The recombinant vectors were called pELP-Cap and pET-Cap (Fig. 1), and the expressed proteins were called ELP-Cap and Cap-His, respectively.
Protein expression
Both pELP-Cap and pET-Cap vectors were transformed individually into BL21 (DE3) E. coli. After growth for 5 h in 2 × YT medium (10 g yeast extract, 16 g tryptone, 5 g NaCl/l, pH 7.2) containing kanamycin (50 μg/ml), the expression of the two fusion proteins was induced for 24 h at 20 °C with 0.2 mM IPTG (isopropyl β-D-thiogalactoside). After 10-min centrifugation at 5000 g, the bacterial pellets were suspended in a lysis buffer (50 mM Tris-HCL, pH 8.0, 2 mM EDTA, 0.5%Triton X-100, 1 mM DTT, 5% glycerol), and disrupted two times at 1300 bar using High Pressure Cell Disruptor (JNBIO, China). After centrifugation for 15 min at 14,000 g, the supernatants were collected for recombinant protein purification.
Protein purification
ELPylated Cap protein was purified by ITC as previously described [18] with slight modification. Briefly, after determination of the transition temperature and salt (NaCl) concentration, ITC was performed in the presence of different concentrations of Triton X-100 and/or different concentrations of urea. After 5-min centrifugation at 14,000 g at room temperature, the protein pellet was washed with 1%Triton X-100 and 0.5 M urea in 3 M NaCl. The residual endotoxin was removed by one round of Triton X-114 isothermal extraction as previously described [19]. His-tagged Cap protein was purified with His-Tagged Protein Purification Kit (CWBIO, China) under denatured conditions by following the manufacturer’s instruction. Finally, the two purified proteins were dialyzed in VLP assembling buffer (50 mM Na2HPO4, 50 mM NaH2PO4, 500 mM NaCl, 1 mM EDTA, 0.03% Tween 80, pH 6.5). After 5-min centrifugation at 12,000 g, the supernatants were collected for further study.
ELP tag cleavage
The recombinant TEV protease was expressed in E. coli as a fusion protein with self-aggregating peptide ELK16 and purified by centrifugation in the presence of 0.5% Triton X-100 as previously described [19]. The purified ELPylated Cap protein (100 μg) was digested overnight with the recombinant protease (30 μg) as previously described [19]. After digestion, the active aggregates of TEV protease were removed by centrifugation and the cleaved ELP tag was removed by one round of ITC as described.
Transmission electron microscopy
Both ELPylated and His-tagged Cap proteins (25 μg) were absorbed onto copper grids (400 meshes) for 2.5 min at room temperature. After drying gently with filter paper, the grids were stained with 3% phosphotungstic acid for 2.5 min. The excess liquid was removed and the samples were observed under transmission electron microscope (Philips, Tecnai 12, Netherland) at an acceleration voltage of 75 kV.
Western blotting
Both ELPylated VLP (ELP-VLP) and His-tagged VLP (VLP) were separated on 12% SDS-PAGE and transferred to nitrocellulose membrane (Merck, USA) using a Mini-Protean® Tetra Cell (Bio-Rad, USA) by following the manufacturer’s instruction. The membrane was blocked for 2 h at 37 °C with 5% skim milk powder in PBST (0.1% Tween 20 in PBS), and incubated for 1 h at 37 °C with home-made pig anti-PCV2 serum (1:500). After three-time washing in PBST, the membrane was incubated for 30 min at 37 °C with DeLyght800-labeled goat anti-pig IgG (1:10,000) (KPL, USA). The hybridization signal was scanned using Infrared Imaging System (Odyssey, USA) at 800 nm by following the manufacturer’s instruction.
Animal immunization and virus challenge
Six-week-old BALB/c mice were purchased from the Center of Comparative Medicine, Yangzhou University. Eighteen mice were randomly divided into three groups (6 for each group). The mice in groups 1 and 2 were immunized intramuscularly with 200 μl (50 μg) of ELP-VLP or VLP without use of adjuvant. The mice in group 3 were injected with the same volume of PBS as the negative control. The primarily immunized mice were boosted with the same dose of antigen at day 14 post immunization (dpi). The blood samples were collected at 7, 14, 21 and 28 dpi for antibody detection. Three mice from each group were sacrificed at 28 dpi for splenocyte isolation and cytokine detection. The remaining three mice in each group were challenged intraperitoneally with 2 × 103 TCID50 (50% tissue culture infectious dose) of PCV2b. On days 7 and 14 post challenge (dpc), the serum samples were collected for PCV2 DNA detection.
Indirect ELISA
Bottom-flattened 96-well microplates were coated overnight at 4 °C with His-tagged Cap protein (5 μg/ml) in 0.1 M carbonate buffer (pH 9.6). After blocking for 1 h at 4 °C with 5% skim milk powder in PBST (0.05% Tween 20 in PBS), the plates were incubated for 1 h at 37 °C with the serum samples (1:100 in PBST). After three-time washing with PBST, HRP (horse radish peroxidase)-conjugated goat anti-mouse IgG (1:10,000 in PBST) (Sangon Biotech, China) was added and incubated for 1 h at 37 °C. After washing again, HRP signal was developed for 20 min with TMB (tetramethylbenzidine) substrate and OD450 values were measured on an ELISA reader.
VN antibody test
The VN antibody test was performed on 96-well microtitration plates using PK-15 cells as the indicator as previously described [20] with slight modification. Briefly, 100 μl (4 × 105) of cells was seeded to each well and grown for 24 h at 37 °C in DMEM containing 10% FBS (fecal bovine serum). Serum samples were heat-inactivated for 30 min at 56 °C, and serially diluted twofold up to 1:512. Each dilution (100 μl) and an equal volume of PCV2b (200 TCID50) were added and incubated for 60 min at 37 °C. After washing with PBS, 200 μl of DMEM containing 2% FBS was added, and incubated for 24 h at 37 °C. After fixing with 80% cold acetone, immunofluorescence was performed using pig anti-PCV2 serum (1:500) as the first antibody and FITC-conjugated goat anti-pig IgG (1:1000) (Sigma, USA) as the second antibody. VN antibody titers were expressed as the highest dilutions in which no or higher than 80% reduction of virus replication was detected as compared to the virus control.
Cytokine detection
Each spleen sample from the immunized mouse was pushed through 1-ml syringe and centrifuged for 10 min at 1500 g. The cell pellet was suspended with 500 μl of PBS and mixed with 3 ml of red blood cell lysis buffer (Beyotime Biotechnology, China). After 5-min incubation at room temperature, the splenocytes were collected by 10-min centrifugation at 1500 g, washed two times with PBS and cultured (5 × 106 cells/ml) overnight in RPMI 1640 medium (Hyclone, USA) supplemented with 10% FBS. After stimulation for 72 h at 37 °C with His-tagged Cap protein (10 μg/ml) or Con A (10 μg/ml) (Sigma, USA), the cell culture was centrifuged for 10 min at 3000 g, and the supernatant was collected for cytokine detection. Interleukin 4 (IL-4), tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) were detected in triplicates using the ELISA Kits (Boster Bio, China) by following the manufacturer’s instruction.
Quantitative PCR
The serum samples from immunized and PCV2-challenged mice were extracted using MiniBEST Viral RNA/DNA Extraction Kit (TaKaRa, China) by following the manufacturer’s instruction. PCV2 DNA copies were detected in triplicates using the Cap-specific forward primer (5-AAGGGCTGGGTTATGGTATG-3) and reverse primer (5-GAGTGGGCTCCAGTGCTGTTA-3). The quantitative PCR (20 μl) was performed using 2 μl of DNA template and SYBR Premix Ex Taq™II (TaKaRa, China) by following the manufacturer’s instruction. The standard curve was generated using pMD-18 T vector (TaKaRa, China) containing the PCR-amplified Cap gene segment.
Statistical analysis
Statistical analysis was performed using SPSS Statistics 22. The results were considered to be statistically significant at p < 0.05 or extremely significant at p < 0.01. For each separate set of data, at least three independent assays were performed and the results were represented as mean ± standard deviation (SD).