Expression and serological application of recombinant epitope-repeat protein carrying an immunodominant epitope of Newcastle disease virus nucleoprotein

Gaikwad, Satish S.; Lee, Hyun-Jeong; Kim, Ji-Ye; Choi, Kang-Seuk

Spodoptera frugiperda 9 (Sf9) cells were cultured at 27°C in Sf-900 II serum free medium (Invitrogen), supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Invitrogen), 50 U/mL penicillin and 50 μg/mL streptomycin. Sf9 cells were transfected with recombinant bacmid DNA using Cellfectin II, a cationic lipid for the transfection of the baculovirus particles according to the manufacturer's instructions. Briefly, for each transfection, 2 mL of Grace's Insect Medium, unsupplemented (without antibiotics and serum) was added in each well, 8×10 5 cells per well were seeded in a 6-well plate and allowed to attach for 2 hours. The bacmid DNA and Cellfectin II (8 μL of reagent) were diluted separately in 100 μL of Grace's medium, unsupplemented (without antibiotics and serum), then mixed and incubated for 30 minutes at room temperature to form lipid-DNA complexes. The cells were washed with fresh medium, and incubated with lipid-DNA complex at 27°C for 5 hours. The transfection solution was removed and 2 mL supplemented Sf-900 II SFM containing 10% FBS was added. Transfected Sf9 cells were incubated at 27°C for 72 hours for baculovirus production. Recombinant baculovirus production was monitored daily by visualization of the cytopathic effects. Three to four days after transfection, recombinant baculovirus was harvested from the cell culture medium and stored at 4°C. Recombinant viruses were identified by polymerase chain reaction using gene vector specific primers (Invitrogen). The resulting baculovirus was passaged three times by infecting more Sf9 cells.