Construction of Recombinant Virus: Insect cell cultures are transfected with wild type Autographa californica nuclear polyhedrosis virus DNA (AcNPV) and baculovirus transfer plasmids and isolates recombinant baculoviruses that arise by homologous recombination. Recombinant viruses in the culture supernatant of the transfected cells are plaque purified by an agarose overlay assay. Individual recombinant viral plaques are then plucked from the agarose, extracted, and used to infect Sf9 insect cells. The infected Sf9 cells (for intracellular proteins) or culture supernatant (for secreted proteins) are provided to the investigator to screen for virus that produces intact protein. Screening is typically done by immunoblot assay or by gene expression.
The investigator is required to clone the cDNA of interest into an appropriate baculovirus transfer plasmid or bacmid. The core does not construct recombinant transfer plasmids, but does provide wild type viral DNA and transfection reagents. Numerous transfer plasmids with convenient restriction sites for cloning are available commercially. We will provide consultation on choice of transfer plasmids.
Amplification and Titering of Recombinant Baculovirus Stocks: After selection of a specific recombinant virus, a passage #1 seed stock is prepared to a 500ml volume and a passage #2 viral stock is tittered as pfu/ml by an agarose overlay plaque assay. Small aliquots of the passage #2 virus are placed at -70°C for long-term storage. The bulk of the amplified 500ml virus stock is stored at 4° C. In addition to amplifying seed viral stocks from newly generated recombinant baculoviruses, the resource also amplifies, titers and stores viral stocks from existing recombinant baculoviruses provided by investigators.
Protein Production in Insect Cells: Protein production is performed on a small scale in conventional spinner culture vessels in the range of 150-1,000 ml volumes or on a large scale in oxygenated 5 liter bioreactors. The resource maintains three insect cell lines for protein production including Sf9, Sf21 and High 5. The Sf9 cells are typically used to express intracellular proteins, while High 5 is often preferable for secreted proteins. The expressed protein is provided to investigators either as a washed cell pellet (intracellular proteins) or as a culture supernatant (secreted protein) after pelleting of cells by centrifugation. Insect cells for protein production are typically grown in Grace's Insect Medium supplemented with 10 percent fetal bovine serum. If requested by an investigator, the insect cells will be adapted to a serum-free defined medium.