The cloning and construction of the expression vector for rPMT-NC and rSly were performed as described previously [11 (link)]. Escherichia coli strain BL21 (DE3) (Invitrogen, CA, USA) harbouring the PMT-NC and Sly recombinant plasmids were cultured in LB broth (Luria–Bertani, Difco, MD, USA) medium and incubated at 37 °C overnight, and the procedures for purification were as previously described.
The construction of a CpG plasmid containing 12 copies of the GACGTT motif is described in the Taiwan patent entitled “DNA adjuvant for waterfowl and live-stock vaccines” (Patent No. I425091), which is available for viewing online through the Intellectual Property Office, Taiwan. Plasmids were amplified in E. coli, purified using a QIAGEN-tip 500 (Qiagen, Hilden, Germany) and dissolved in sterile PBS for formulation. The purified rPMT-NC (200 μg/mL) as an antigen was emulsified with 50% water-in-oil-in-water (w/o/w) adjuvant (ISA206, Seppic, France), CpG plasmid (200 μg/mL) or rSly protein (100 μg/mL) and stored at 4 °C. The sterility of the vaccines was confirmed by culture with trypticase soy agar (Difco, MD, USA) (TSA, 37 °C), thioglycollate agar (Difco) (TGC, 37 °C) and Sabouraud dextrose agar (Difco) (SDA, 25 °C). All vaccine dosages had a final volume of 2 mL.
The construction of a CpG plasmid containing 12 copies of the GACGTT motif is described in the Taiwan patent entitled “DNA adjuvant for waterfowl and live-stock vaccines” (Patent No. I425091), which is available for viewing online through the Intellectual Property Office, Taiwan. Plasmids were amplified in E. coli, purified using a QIAGEN-tip 500 (Qiagen, Hilden, Germany) and dissolved in sterile PBS for formulation. The purified rPMT-NC (200 μg/mL) as an antigen was emulsified with 50% water-in-oil-in-water (w/o/w) adjuvant (ISA206, Seppic, France), CpG plasmid (200 μg/mL) or rSly protein (100 μg/mL) and stored at 4 °C. The sterility of the vaccines was confirmed by culture with trypticase soy agar (Difco, MD, USA) (TSA, 37 °C), thioglycollate agar (Difco) (TGC, 37 °C) and Sabouraud dextrose agar (Difco) (SDA, 25 °C). All vaccine dosages had a final volume of 2 mL.