Materials
Spodoptera frugiperda (Sf9) cell lines were grown and maintained at 27℃ in SF900Ⅲ medium (Invitrogen Corporation, USA). Plasmid pET28a-CPV-VP2 containing CPV vp2 gene (GenBank: MK518021.1) and Rabbit anti-VP2 polyclonal antibody were kindly provided by Dr. Qinghai Tang[15]. The improved baculovirus expression system including the vector pYBDM-IM (an IRES drived mcherry fragmemnt is insert into the sphI and kpnI sites of pFBDM), Esherichia coli AcMultiBacmid/rSW106/asd-/inv+ were constructed as described previously [14].
Construction Of Recombinant Transfer Vectors
To facilitate expression of the VP2 genes in insect cells, full-length vp2 gene was amplified by PCR from plasmid pET28a-CPV-VP2. The primers used for vp2 gene amplification were VP2 Forward primer (5’-GGATCCCGGGATGAGTGATGG AGCAGT-3’ containing one site of BamHI and SmaI each) and VP2 Reverse primer (5’-TCTAGAGTCGACTTAATATAATTTTCTAG-3’ containing one site of XbaI and SalI each). The PCR product encoding VP2 ORF was treated with BamHI/XbaI and cloned into the multiple cloning sites (MCS) under the polyhedrin promoter, the resultant plasmid was designated pYBDM-IM-ph-VP2. Similarly, the amplified VP2 fragment was inserted into SmaI/XhoI restriction sites, downstream from the p10 promoter of the vector pYBDM-IM or pYBDM-IM-ph-VP2, to generate the plasmid of pYBDM-IM-p10-VP2 or pYBDM-IM-2VP2 respectively (Fig. 1). All constructs were confirmed by DNA sequencing.
Generation Of Recombinant Bacmids
Individual recombinant plasmids were transformed into E. coli AcMultiBacmid/rSW106/asd−/inv+ competent cells, allowing the transposition of CPV VP2 gene to Bacmid between the mini-Tn7 element on the transfer vector and the mini-att Tn7 target site on the Bacmid to generate the recombinant bacmids. The obtained recombinant bacmids were characterized by white–blue and PCR screening using vp2 forward primer and M13 reverse primer to confirm the insertion of the VP2 gene.
Production Of Recombinant Baculoviruses
E.coli AcMultiBacmid/rSW106/asd-/inv + cells with recombinant VP2 genes were grown in LB broth supplemented with 0.5 mM DAP, 10 µg/ml tetracyclines, 7 µg/ml gentamicin, 25 µg/ml spectinomycin and 50 µg/ml kanamycin at 30 °C, The overnight culture were collected by centrifugation (3000 × g) and resuspended in distilled ultrapure water for three times. The pellet was resuspended in SF900Ⅲ medium and adjusted to different densities (105-108 cells/mL). Sf9 cells at 105/ml were incubated overnight in 24-well plates (70–80% confluent single layer). After removing the medium, 500µL bacterial cells at different concentrations were added to each well to infect sf9 cells. After culturing at 27 °C for 4–5 h, each well was washed three times. 500µL fresh SF900Ⅲ medium was then added and incubated for 3–5 days. When the mCherry fluorescence was observed using reverted fluorescence microscopy (507 nm excitation), indicating that sf9 cells were infected successfully. The supernatant containing recombinant baculovirus was harvested and infected again with sf9 cells. Titers of the baculovirus were determined by a plaque assay.
Production And Purification Of Cpv Vp2 Protein
2 × 108 of sf9 insect cells were grown in 100 mL volumes in 500 ml baffled glass flasks and incubated in a rotary shaker at 80 rpm and 28 °C. The initial cell density was 2.0 × 105cells/mL. The cultures were infected with the recombinant baculoviruses, Ac-IM-ph-VP2, Ac-IM-p10-VP2 and Ac-IM-2VP2 at a multiplicity of infection (MOI) of 5. At 96 hours post infection (h.p.i.), the cells were harvested by centrifugation (1000 × g, 10 min, 4 °C) and lysed by sonication. The lysed Sf9 cells were centrifuged at 12,000 × g for 10 min, the supernatant was purified using Ni-NTA agarose according to the manufacturer’s instructions. The concentration of VP2 protein was measured with the His Tag ELISA Detection Kit (GenScript).
Western blot analysis of recombinant CPV VP2 protein
The expression of CPV VP2 proteins was determined by Western blot assay. The supernatants of samples, which were purified as described above, were separated by 12% SDS-PAGE and transferred onto polyvinylidene fluoride membranes and then blocked with 10% skimmed milk for 2 h. After five washes with PBST (PBS plus 0.05% Tween-20) for 5 min each time, the membranes were incubated overnight with mouse anti-His monoclonal antibody (1: 5000 dilution) and rabbit anti-VP2 polyclonal antibody (1: 200 dilution) at 4 °C, respectively. After five washes with PBST for 5 min each time, the membranes were incubated with HRP-conjugated goat anti mouse IgG antibody (1: 2000 dilution) and HRP-conjugated goat anti rabbit IgG antibody (1: 2000 dilution), respectively. After five washes with PBST for 5 min each time, detection was performed with DAB (3, 3’-diaminobenzidine) solution (Boshide, Wuhan, China).
Animals Experiment
The purified VP2 proteins were adjusted at a final concentration of 1 mg/mL. All animal protocols were performed in accordance with the guidelines of the ethical committee of Nanyang Normal University. The mice were purchased from Wuhan Biological products Research Institute Co., Ltd. Fifteen BALB/c mice (6-week-old, female) were randomized into three groups (n = 5). Mice in group A were intramuscularly injected with 100µL of purified VP2 protein mixed with Freund’s adjuvant. Mice in group B was injected with 100µL commercial live-attenuated vaccine as a positive control. Mice in group C were injected with 100µL/mouse of PBS as a negative control. Mice from all groups were injected 2 times at 2-week intervals (Days 0 and 14). Blood samples were collected from the forelimb veil at 0, 7, 14, 21 and 28 days post-vaccination (dpv).
Hemagglutination Inhibition (hi) Test
Serum samples from mice were inactivated at 56 °C for 30 min, then serially diluted two-fold (25µL of serum) in V-96-well plates. Subsequently 25µL 4 hemagglutination units of CPV were added. The mixture were incubated at 37 °C for 1 h after which 50µL 0.8% pig erythrocytes were added. Hemagglutination inhibition antibody titers were expressed as the reciprocal of the highest serum dilution that completely inhibited hemagglutination.
Statistical analysis
The experimental data were analyzed using a one-way analysis of variance (ANOVA), combined with Tukey’s post hoc test. P < 0.05 was considered statistically significant.