Normas de diseño y planificación

For the production of recombinant proteins in eukaryotic cells the baculovirus expression system was used. This system is based on the generation of baculovirus particles carrying the target gene in their chromosome and the subsequent infection of insect cells with these virus particles. The protocols and procedures used and described here for the generation of recombinant virus particles, growth and maintenance of insect cell cultures and recombinant protein production were derived mainly from the following user manuals provided by the manufacturer (Invitrogen™):

Growth and Maintenance of Insect Cell Lines (Version K)

Baculovirus Expression System with Gateway® _{Technology (Version E)} Bac-to-Bac® _{Baculovirus Expression System (Version E)}

The following figure indicates the main steps involved in the production of a target protein using the BVES with Gateway® _Technology.

Figure 5.2: Experimental outline for the production of a recombinant target protein using the BVES with Gateway® _Technology.

(adapted from „Bac-to-Bac® _{Baculovirus Expression System‟ User Manual, Version E,}

Sf9 cells that are pre-adapted to serum-free medium and suspension culture methods were used throughout these experiments. To avoid bacterial and fungal contamination all experiments were carried out under sterile conditions in a biohazard cabinet that had been exposed to at least 30 minutes of UV radiation and sprayed with a 70% (v/v) ethanol solution. All equipment that was used in the biohazard cabinet during the experiments was sprayed with a 70% (v/v) ethanol solution just before transfer to the cabinet. Disposables (tips, microcentrifuge tubes etc.), bottles and Erlenmeyer flasks were autoclaved twice before use.

5.2.6.1 Initiation and Maintenance of Spodoptera frugiperda (Sf9) cells

For initiation of Sf9 cells from frozen stocks, a vial containing 1 mL of 1.5 10 Sf9 cells was removed from liquid nitrogen storage and quickly transferred to a 37°C water bath until almost thawed. The cell suspension was then transferred to an Erlenmeyer flask containing 27 mL of pre-warmed serum-free medium Sf-900 II SFM. The flask was transferred to a 27°C incubator and incubated under gentle orbital shaking at 150 rpm until the cell density reached > 2 106 _{cells . Cells were then subcultured by} seeding flasks with 3-5 105 _{viable cells mL.}

Stock cultures were maintained as 50 mL cultures in 250 mL Erlenmeyer flasks. These cultures were grown until cell density reached 2-3 106 _{viable cells mL and then diluted into fresh medium to a density of} 3 105 _{cells mL. This subculturing procedure was generally performed twice} weekly to maintain cells in optimal condition.

The cell density was determined using a haemocytometer. Cell viability was determined by adding 0.1 mL of a 0.4% trypan blue solution to 1 mL of culture. Cells that take up the stain, and therefore appear blue, are considered as not viable. Cell viability was calculated as number of viable cells divided by the total number of cells within the haemocytometer grid.

5.2.6.2 Transfection of Sf9 cells and Preparation of Viral Stocks

Bacmid DNA to be used for transfection of insect cells was purified using a PureLink® _{HQ Mini Plasmid Purification Kit, which produces DNA free from} contaminants that might interfere with the transfection reagent and so decrease transfection efficiency. Cells used for transfections were in a range of 1.5-2 106 _{cells mL at a viability of ≥95%.}

Transfections were performed in 6-well plates (1 well/bacmid) under the following general conditions:

Number of cells 9.0  105 _cells/well

Bacmid DNA 1.0-2.0 µg

Cellfectin® _{II reagent 6.0 µL}

The cells were added to 2 mL Grace‟s Medium (unsupplemented) and allowed to attach for ~30 minutes at room temperature. Cellfectin® _{reagent and} bacmid DNA were each mixed separately with 100 µL Grace‟s Medium (unsupplemented). These mixtures were then combined, mixed gently and incubated for 15-30 minutes at room temperature. After the incubation, the DNA-Cellfectin® _{mixture was added slowly to the cells and the plates incubated} at 27°C for 3-4 h. The transfection mixture was then removed from the cells and replaced with 2 mL Sf-900 II SFM. Cells were incubated at 27°C for 3-5 days until signs of infection became visible. The medium was removed from the cells, centrifuged to remove cells and cell debris and the clarified supernatant kept at 4°C in the dark as P1 viral stock.

Amplification of the P1 viral stock was carried out in a 10 mL suspension culture at a cell density of 2 106 _{cells mL. Cells were infected with 0.4 mL P1} viral stock (multiplicity of infection (MOI; ratio virus:Sf9 cells) of ~ 0.1) and incubated for 48-72 h at 27°C. Cells and cell debris were removed by centrifugation and the supernatant (= P2 viral stock) was stored at 4°C in the dark.

5.2.6.3 Determination of Virus Titres - Plaque Assay

The determination of the P2 viral stock titre was performed in 6-well plates, with 2 plates being required for every stock to be titered. 2 mL of a 5  105 cells/mL suspension were transferred into each well and the plates incubated at room temperature for 1 h. The medium was subsequently replaced with 1 mL of a serial dilution (in SF-900 II SFM) of the P2 viral stock to be titered. Dilutions used were 10-4 _{to 10}-8 _{and a negative control containing no virus was included in} each plate (each with duplicate). Following 1 h incubation at room temperature to allow infection of the cells, the stock dilutions were replaced with 2 mL of plaquing medium. The plates were left for 1 h at room temperature to allow the agarose overlay to harden and then moved to a 27°C humidified incubator where they were incubated for 7-10 days until plaques were visible. Plaques were visualised by staining with neutral red and counted. The following formula was used to calculate the viral titre:

Plaquing medium (volumes per assay): Sf-900 SFM (1.3×) 30.0 mL

4% Agarose, melted 10.0 mL

The medium was equilibrated at 40°C prior to use.

The P2 viral stock was used for infection of Sf9 cells in protein expression trials and expression optimisation experiments.

5.3

Results & Discussion