Evaluation on the Efficacy and Immunogenicity of Recombinant DNA Plasmids Expressing Spike Genes from Porcine Transmissible Gastroenteritis Virus and Porcine Epidemic Diarrhea Virus

Meng, Fandan; Ren, Yudong; Suo, Siqingaowa; Sun, Xuejiao; Li, Xunliang; Li, Pengchong; Yang, Wei; Li, Guangxing; Li, Lu; Schwegmann-Wessels, Christel; Herrler, Georg; Ren, Xiaofeng

Plasmid DNAs pIRES-(TGEV-S1-PEDV-S1), pIRES-(TGEV-S1-PEDV-S) and pIRES were chromatographically purified from bacterial lysates (Qiagen, Germany), precipitated, and then washed with ethanol. All plasmids were dissolved in 0.1 M PBS to a final concentration of 1 mg/ml. For immunofluorescence assays, BHK-21 cells from American Type Culture Collection (ATCC) were cultured to 90% confluency in 6-well plates at 37uC (approx 24 h) then transformed with 3 mg of pIRES-(TGEV-S1-PEDV-S1) and pIRES-(TGEV-S1-PEDV-S), or pIRES using lipofectamine 2000. The next day, indirect immunofluorescence assays were performed with modifications as described [22, 24, 37] . After fixation by formalin, the cells were incubated for 20 min with 0.2% Triton X-100. Followed by incubation for 1 h with polyclonal antisera (1:200 dilution in 1% BSA) against PEDV (prepared in our laboratory) or TGEV (prepared in our laboratory). After washing with PBS, the cells were incubated in the dark with FITC-labeled goat anti-rabbit IgG (1:500) for 1 h. The green fluorescence signals were analyzed by fluorescence microscopy (Leica, Germany). For Western blot, 6 mg of each plasmid were transformed into BHK-21 cells in 6-well plates. BHK-21 cells transfected with empty vector were used as control. After 24 hours, cells were treated with 1% Triton X-100 and centrifuged at 12,000 rpm for 5 minutes, respectively. Then the supernatants were subjected to 10% SDS-PAGE. The proteins were electronically transferred to a nitrocellulose (NC) membrane. The NC membrane was blocked overnight at 4u using 5% non-fat dry milk in PBS-0.05% Tween-20 (PBST) followed by incubation with the anti-TGEV or anti-PEDV serum (1:300 dilution in PBST) at 37uC for 1 h. The membrane was incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (1:2000 dilution in PBST) at 37uC for 1 h, after complete washing with PBST. The protein bands were visualized using chemiluminescence reagents (Roche, Switzerland).