Baculovirus Generation and Amplificaiton

Generation of Baculovirus

1. Subclone gene into pFastBac-DUAL
2. Select and isolate the recombinant donor plasmid.
3. Transform the recombinant donor plasmid into DH10Bac E. coli and select the recombinant bacmid.
4. Seed 9 × 105 Sf-9 cells in 2 mL insect cell medium to each 35-mm dish.
5. Transfect the recombinant bacmid into Sf-9 cells
6. After transfection, wash the Sf-9 cells with 2 mL medium and continue to culture the cells at 27 °C.
7. Harvest the recombinant virus in the culture medium at 72 h posttransfection. Determine the virus titer

Amplification of Baculovirus

1. Seed Sf-9 cells to T-25 flasks at ~50–70% confluency and allow the cells to attach at 27 °C for at least 1 h.
2. Remove the medium and inoculate the virus at a low MOI. Replenish complete medium to a final volume of 0.5–1 mL.
3. Gently shake the flask on a rocking plate at room temperature for 1 h.
4. Add complete medium to a final volume of 5–7 mL. Incubate in a 27 °C humidified incubator.
5. Collect the medium at 72–96 h postinfection and centrifuge at 1,000 × g for 5 min.
6. Collect and store the virus supernatant at 4 °C, protected from light. Store at –80 °C for long-term storage.
7. To amplify the virus stock to a larger scale, the process can be repeated by infecting the cells cultured in larger spinner flasks at a low MOI.
8. If concentrated baculovirus is required, load 33 mL virus supernatant in each of six 38 mL ultracentrifuge tubes.
9. Underlay with 3 mL sucrose cushion solution to each tube.
10. Centrifuge at 80,000 × g (24,000 rpm in an SW28 rotor) for 75–90 min at 4 °C.
11. Decant the supernatant and carefully remove traces of sucrose. The viral pellet appears translucent white.
12. Resuspend the pellets in 1–2 mL PBS (pH 6.2).
13. Filter the concentrated virus through 0.22 μm filters and determine the virus titer. Store at 4 °C.