Baculovirus Amplification

High titer recombinant virus stocks (~ 2 x 108pfu/ml) are used for infection of cells at optimal Multiplicity Of Infection (MOI = # of virus / # of cells) resulting in maximum protein production. Prepare large stocks of virus by infecting insect cells at a low multiplicity of infection (MOI <1) and harvesting supernatant 3-5 days post infection (pi). It is critical to use a low MOI because passaging the virus at high MOI increases the possibility of virus with mutations in their genome. The number of mutant virus is also increased by serial passage; it may be advantageous to maintain a low-passage seed stock from which larger working stocks are amplified. Eventually, the titer in the seed stock will be reduced through storage, and it becomes necessary to generate a new passage seed stock.

Since recombinant virus generated using BD BaculoGold™ are greater than 99% of the total virus population, it may not be necessary to initially prepare all stocks from a clonal viral population. However, if there is a reduction in protein production after multiple passages of a viral stock, it may be necessary to isolate clonal viral populations by end-point dilution assay (EPDA) or plaque purification. After verification of protein production, the clonal virus population can be amplified to produce a high titer stock. The viral stock is then ready for large scale protein production.

Protocol

  1. Seed 2 x 107Sf9 cells on a 15 cm plate (BD Falcon™, Cat. No. 352097). Allow cells to attach for 15 min, confirm ~ 50% confluency by visualization using a light microscope. Change to fresh TNM-FH medium.
  2. Add 100 µl -1 ml of your low titer recombinant stock to the plate. If you know the virus titer of your stock solution, use MOI < 1. Repetitive infections with an MOI > 1 may select for mutants which no longer express your gene.
  3. Incubate the cells at 27°C for 3 days. Check for signs of infection 2 days after the start of infection.
  4. Harvest the supernatant from the plate. Remove cellular debris by centrifugation at 10,000 x g.
  5. Store the virus supernatant in a sterile tube at 4°C for up to six months. For longer storage periods, virus supernatant should be frozen in aliquots at -80°C.
  6. Determine the virus titer of your amplification solution using the plaque assay procedure or the modified end-point dilution assay. Amplification typically is done 2 or 3 times to attain a high viral titer (~2 x 108/ml).

Storing Virus Particles

Supernatants containing baculovirus particles may be stored in a sterile tube at 4°C for up to 6 months or frozen, in aliquots, at -80°C for a longer period of time. If frozen, avoid multiple freeze and thaw cycles. Upon freezing, the viral titer may decrease and should be re-amplified when thawed. Store viral stocks in the dark; titers appear to decrease when exposed to fluorescent light for prolonged periods of time. The best way to preserve a recombinant virus is to isolate its DNA and store it at -80°C.

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