Protocol

Abbreviation

WU_baculovirus_expression_1

Name

WU_baculovirus_expression_1

Laboratory name

Washington University

Type

selection

Description

Recombinant baculovirus were generated with flashBAC baculovirus expression system (Mirus). Basically, the ectodomain of HA was cloned into a modified pOET1 vector (see cloning method) and co-transfected with flashback DNA into sf9 insect cells cultured in 12-well plates. The medium was SFIII. After 5 days incubation, we harvested the culture medium containing recombinant virus. This is the P0 virus seed stock. Then, we amplified P1 and P2 virus in sf9 cells cultured in 200 ml suspension SFIII medium at a density of 2 x 106 cells/ml. Typically, P2 virus were used for protein expression. Hi5 cells were infected at a density of 2 x 106 cells/ml with P2 virus at MOI of 1-5 and the supernatant was harvested after 72hr post-infection. Express Five serum free meduim was used for protein expression. Cells were grown in baffled shake flasks at 100 rpm in a humidified 28 °C incubator (without CO2).