The recombinant bacmid DNA was then used to transfect Sf9 cells grown in Grace’s medium. At a density of 1X106 cells per 35-mm well, the cells were transfected with the bacmid DNA using Cellfectin reagent (Invitrogen) according to the manufacturer’s protocol. The recombinant virus was harvested at 72 h posttransfection. According to the manual of the kit, plaque assays were performed to determine the titer of recovered recombinant virion particles, and each recombinant viral stock was made for expression studies.
Production of Recombinant Protein
To analyze the production of the rgLh, Sf9 cells were grown in 75-mm tissue culture flasks until 70%-80% confluency was reached. The cells were infected with recombinant virus at 10 multiplicities of infecton (moi) and the infection was carried out at 27°C for 0 h, 24 h, 48 h, 72 h, 96 h, and 120 h. To examine the intracellular production, cytoplasmic extract was prepared from the infected cells as described by Mathur et al.. Briefly, cells were washed with phosphate-buffered saline and suspended in 0.5 ml of homogenization buffer containing 10 mM Tris-HCl (pH 8.0) and 25 mM NaCl, after which they were disrupted with a Dounce homogenizer.