Baculovirus-Mediated Expression of IgT

The full-length IgT gene of ASB was amplified with primers IgT NotI F and IgT XhoI R (Table S1) and inserted into pFastBac HT A transfer vector. The construct was integrated into the baculovirus genome with DH10BAC™ and the Bac-To-Bac system (Invitrogen). The bacmid DNA with IgT gene was purified for transfection into Spodoptera frugiperda (Sf9 III) cells (ATCC) for producing recombinant baculovirus (Bac-IgT). The Sf9 III cells were grown at 27°C in serum-free medium SF-900 III (Invitrogen). Procedures for the generation of recombinant baculovirus were carried out according to the manufacturer’s instructions (Invitrogen). Briefly, 1.2 X 10⁶ Sf9 III cells were plated onto 6-well plates for 1 h. After attachment, 4 µg of recombinant bacmid DNA Bac-IgT and 10 µl Cellfectin II (Invitrogen) were diluted with culture medium and used for transfection of Sf9 III cells. Transfection was performed for 5 h at 27°C with the replacement of fresh SF-900 III medium after incubation. Transfected cells were kept at 27°C for 72 h. Following incubation, the supernatant containing recombinant viruses (Bac-IgT) was utilized for infection of fresh Sf9 III cells. For large scale viral production, Sf9 III cells were infected in suspension cultures of 2×10⁶ cells/ml with the collection of supernatant four days post-infection.