Cells and virus
PK-15 cells (ATCC™ CCL-33) were routinely cultured in Dulbecco’s Modified Eagle Medium (DMEM; Gibco) containing 10% fetal bovine serum (FBS; HyClone), 100 IU/mL Penicillin and 100 mg/L Streptomycin (InvivoGen, France) at 37 °C in a 5% CO2 atmosphere. PCV2 strain DF-1 (GenBank accession: JN119255) was grown in PK-15 cells and utilized for virus neutralization assay (NA) and challenge experiment.
Experimental animals
Thirty-six female BALB/c mice of 4 weeks old were purchased from the Experimental Animal Center of Zhengzhou University. The experimental mice were separated in six groups and given five days to acclimate the housing environmental conditions (temperature: 22 ± 3 °C, humidity: 55 ± 15%, lighting: 12 h light/dark cycle). The mice were allowed free access to clean water and food. The animal experiments were carried out according to the Animal Experiment Committee of Henan Academy of Agricultural Sciences (Approval number SYXK 2014-0007). All animals received humane care in compliance with good animal practice according to the animal ethics procedures and guidelines of China. All sections of this report adhere to the ARRIVE Guidelines for reporting animal research [16]. A completed ARRIVE guidelines checklist is included in ARRIVE Guidelines Checklist S1.
Plasmids construction
As shown in Fig. 1a, complete Cap gene which sources from PCV2 strain (GenBank Acc. No. AY686763) bound to the truncated calreticulin (120-308 aa/120-250 aa) (GenBank Acc. No. EU639407) at N/C terminal using 4×GGGGS or 5×GGGGS linker. All these four recombinant fragments, named rP4C/rC4P/rP5F/rF5P, were synthesized after codon optimization by Genscript. All the plasmids were inserted into pEG-28a in BamHI and XhoI sites and then transformed into E. coli BL21 (DE3) competent cells, respectively.
Proteins expression and purification
All positive clones were selected and cultured in Luria-Bertani (LB) medium with 50 mg/L kanamycin and then induced with 0.1 mM IPTG at 37 ℃ for 6 h. The parameters of protein expression were optimized according to IPTG concentrations (0.1mM, 0.2mM), and the induction temperature and time (18 °C for 24 h, 25 °C for 16 h). Protein expression was verified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The optimal harvest cells were suspended in lysis buffer (50 mM PB, 150 mM NaCl, 5% (w/v) Glycerol, 5% (w/v) Triton X-100, 2 mM EDTA, 2 mM DTT, pH 7.0) and then lysed by sonication (99 cycles of 2 s On/5 s Off, amp 25 %)). After centrifugation, the precipitation was removed and supernatant of rF5P was purified by Ni-NTA affinity chromatography. After washing the Ni-NTA column (Invitrogen, USA) with wash buffer (50 mM PB, 150 mM NaCl, 30 mM imidazole, pH 7.0), the rF5P was eluted with elution buffer (50 mM PB, 150 mM NaCl, 250 mM imidazole, pH 7.0). Fractions were analyzed by SDS-PAGE.
The purified rF5P was enriched and analyzed by size-exclusion chromatography with Superdex 200 prep grade (pg) (26/60) gel filtration column (GE Healthcare, USA). The samples were eluted using lysis buffer at a flow rate of 1 mL/min and detected at 280 nm wavelength. The collected fractions were identified by SDS-PAGE and Western Blot and then quantified using BCA Protein Assay Kit (TIANGEN, China).
Characterization of rF5P
The enriched purified rF5P was detected under a transmission electron microscopy (TEM) using the negative staining method and dynamic light scattering (DLS) according to the previous study [17].
Antigenicity analysis of rF5P
Indirect enzyme-linked immunosorbent assay (ELISA) was performed to test the antigenicity of rF5P with swine clinical positive/negative serum and mouse anti-PCV2 monoclonal antibodies (mAbs) 6A4 (Abcam, USA). The ELISA procedure was operated as routine.
Vaccination and challenge in mice
Thirty-six female BALB/c mice of 4 weeks old were divided randomly into 6 groups (n= 6). The mice were inoculated subcutaneously with 30 μg and 15 μg of rF5P as Group rF5PH and Group rF5PL, respectively; 50 μL of commercial inactivated Circovac® vaccine (Merial), subunit vaccine Ingelvac CircoFLEX® (Boehringer Ingelheim) and PBS were classified as positive and negative controls, named as Group MLY, BLG and PBS, respectively.
The rF5P was diluted in 50 μL of PBS and then emulsified with 50 μL of Complete Freund's adjuvant for the first immunization, and subsequently with 50 μL of Incomplete Freund's adjuvant for booster at an interval of 4 weeks. At 56 days after the first immunization, 3 mice from each group were sacrificed by cervical dislocation for both lymphocyte proliferation assay and cytokine production. The rest alive mice received 100 μL of 106.5 (TCID50)/mL PCV2 DF-1 strain, and they were monitored for 28 days. Next, the mice were sacrificed for PCV2 content in different organs. Blood samples were collected from the tail veins each week.
Antibody response in mice
The serum samples taken at each point post immunization were monitored using Porcine circovirus type 2 ELISA antibody test kit (KeQian, China). Operation steps followed the product manual.
The abilities of all serum samples to neutralize the PCV2 strain DF-1 were assessed using Virus neutralization assay (NA). In brief, 50 μL sera pre-treated at 56 °C for 30 min were diluted in a serial two-fold way from 1:2 to 1:1024 and mixed with an equal volume of virus (400 TCID50) at 37 °C for 1 h. The serum-virus complex was transferred into confluent PK-15 cells in each well and then incubated at 37 °C for 72 h. Since no visible cytopathic effect was verified, immunoperoxidase monolayer assay (IPMA) was performed to ascertain the presence of the virus [11]. Virus neutralization titer was expressed as the highest dilution as log2NA in which no higher than 80% reduction of virus replication was detected as compared with the virus control.
Spleen lymphocyte proliferation assay
Spleens of mice from each group were removed at 56 days post inoculation (dpi). The spleen lymphocytes were isolated by Lydroxypropylmethyl Cellulose (Solarbio, China) and then resuspended in RPMI 1640 medium containing 10% FBS. Lymphocyte proliferation assay was performed by cell counting kit-8 assay (Beyotime Biotechnology, China) as previously described [18]. T lymphocyte proliferation was represented as the stimulation index (SI), the ratio of the mean reading of stimulated wells to unstimulated ones.
Analysis of cytokine production by activated lymphocytes
The supernatants from the spleen lymphocytes employed in the proliferation assay were removed and adopted to analyze cytokines. The assays were performed using commercially available mice IFN-γ, IL-10, IL-18, TNF-ɑ and GM-CSF ELISA kits (USCN Life Science, China) following the manufacturer’s instructions.
Determination of PCV2 in tissue
PCV2 DNA from different organs (heart, liver, spleen, lung and kidney) of all groups at 28 days post-challenge was quantified by real-time fluorescent quantitative PCR as previously described [19]. The viral load was calculated according to the standard curve plotting Ct values against different dilutions of a standard plasmid.
Statistical analyses
GraphPad Prism version 5.00 (USA) analysis of variance (ANOVA) was performed. The data is expressed as the mean ± SEM. Statistical significance was found by two-way or one-way ANOVA at*P < 0.05, **P < 0.01, ***P < 0.001; ns represents no statistical significance.