Antecedentes

2.1.3.1 Small scale double-stranded plasmid DNA preparation

Plasmid DNA preparations yielding 5-20|Xg plasmid DNA were performed by a (modified Alkali Lysis) method derived from Sambrook et al. (pl.25-1.28, 1989). A 5ml culture in LB broth supplemented with 50ug/ml ampicillin was shaken at 37°C for

6-16hrs. Cells were pelleted (5000g, lOmin) then resuspended in 200)J.l GTE (50mM glucose, 25mM Tris-HCl pH 8.0, lOmM EDTA) and transferred to 1.5ml microcentrifuge tubes. Freshly prepared 0.2M NaOH/1% SDS (300|xl) was added, the tube inverted several times, incubated on ice for 5min and neutralized by the addition of 300|il of 3.CM potassium acetate pH4.8. The tube was mixed gently, incubated on ice for 5min and centrifuged at lOOOg for lOmin at room temperature to remove the cellular debris. The supernatant was treated with 20|ig RNase A at 37°C for 20min, then extracted twice with 400|xl of chloroform. DNA was then precipitated by adding an equal volume of isopropanol and centrifugating at lOOOOg for lOmin at room temperature. The DNA pellet was washed with 70% ethanol, dried under vacuum, then dissolved in 32pl water. DNA was further purified by PEG precipitation by adding 8.0|il of 4M NaCl and 40fil of sterile 13% PEG8000. After thorough mixing, the sample was incubated on ice for 30min and centrifuged at 10,000g for 15min at 4°C. The pellet was washed with 70% ethanol, dried under vacuum, resuspended in 20|il of water and stored at -20°C. The quality of DNA prepared by this method was found to be suitable for DNA sequence analysis.

2.1.3.2 Large scale double-stranded plasmid DNA preparation

Large scale double stranded plasmid DNA preparations or 'maxi-preps’, to provide 0.1- 2mg plasmid DNA for transfection or digestion and use in the construction of other plasmids, were performed using the Wizard Maxi prep kit (Promega) according to the manufacturer’s instructions. The concentration of plasmid was assessed by agarose gel electrophoresis and comparison of intensity of ethidium bromide staining. DNA was to be used for transfection of mammalian cells, was purified using an additional extraction with phenol, followed by another chloroform extraction.

2.1.4 DNA Sequencing

DNA sequencing was performed by the dideoxy chain termination method (Sanger et al., 1977). DNA prepared as in section 2.1.3.1 was sequenced using the ABI PRISM™ Ready Reaction DyeDeoxyTM Terminator Cycle Sequencing Kit according to the manufacturer’s instructions. This method uses four dideoxy nucleotides, G, A, T, and C, labelled with different fluorescent dyes. These dideoxy-nucleotides terminate PCR extension of template DNA, which is carried out using AmpliTaq DNA polymerase (Perkin-Elmer). PCR amplification is carried out for 25 cycles of dénaturation (96°C, 30s), annealing (50°C, 30s) and extension (60°C, 4min). PCR products were resolved by electrophoresis on a 6% acrylamide gel (Sequagel-6, National Diagnostic) under denaturing conditions. The gel was electrophoresed for 12hrs at 30 watts and 1280 volts on a model 373 automated sequencer (Applied

Biosystems). DNA sequence assembly and analysis was performed using the AUTOASSEMBLER SEQUENCE NAVIGATOR software (ABI).

2.2 Cell Culture Methodology

2.2.1 General cell culture techniques and seeding densities

for insect cells.

Spodoptera frugiperda (Sf9) cells were maintained in culture as previously described in (Smith and Johnson, 1988). All culture media were warmed to 27°C prior to use and the cells were routinely incubated at 27°C. The culture medium used throughout was IPL41 (Gibco.BRL) supplemented with 10% (v/v) PCS, 2% yeastolate and 1% lipid supplement (Gibco). Routine culture was performed in the absence of antibiotics although 1% (v/v) fungizone and 50pg gentamycin (Gibco.BRL) were added during amplification of virus stocks. When growing monolayer cultures, 80-90% confluent cells were routinely subcultured in a 175cm^ flask (Sf9 cells have a doubling time of 18-24hrs. Cells were grown to a density of 3-4x1 O^/ml and diluted 1/10). Cells were detached by gentle agitation, then diluted 1:2 to 1:40 into a new flask containing fresh IPL41 media in a final volume of 20mls. The flask was rocked gently to distribute the cells evenly, then incubated at 27°C. For large scale protein production, Sf9 cells were grown in suspension. Suspension cultures were started at an initial density of 5x10^ cells/ml. They were incubated at 27 °C with constant stirring at 50-60 ipm and required subculturing every 72hrs when the cell density reached 3-4x10^ cells/ml.

2.2.1.2 Transfection of Sf9 cells.

The insertion of a gene of interest into the baculovirus genome is usually achieved by homologous recombination between a transfer plasmid and the viral DNA. This occurs when the plasmid and viral DNAs are simultaneously introduced (co-transfected) into host insect cells. A liposome-mediated transfection was employed (O'Reilly, 1994).

Cells were seeded (3xlO^/5ml) into 5ml IPL41 medium in 25 cm^ flasks and allowed to attach for at least Ihr. The lipofectin solution (GibcoBRL) was diluted 2:1 with sterile water and then mixed with an equal volume of a 1:6 mixture of BaculoGold DNA (Pharmingen) together with the recombinant plasmid in a polystyrene tube. The mixture was incubated at room temperature for 15min to allow the liposomes and DNA to fuse. Meanwhile, the cells were washed twice in serum and lipid free IPL41, and the transfection mixture was then added to the cells in 1.5 mis of serum free IPL41 and incubated overnight, after which time the transfection medium was replaced with complete IPL41. Medium containing recombinant virus was harvested after 4-5 days.

2.2.1.3 Baculovirus production and amplification

The virus produced as described in section 2.2.1.3 was harvested by centrifugation at 3000g and 1ml aliquots were used to infect 3x10^ Sf9 cells 75 cm^ flasks. Four days post-infection, virus was harvested and 1ml of this virus stock was used to infect 2x10^ cells in a 175cm^ flask. This amplified virus was then used to make a passage 4 virus stock which was titred to determine the amount of virus required for optimum protein production. A range of dilutions of the virus stock, generally between 1/5 and 1/10,000 were used to infect 1x10^ cells in 25cm^ flasks. Cells were harvested at 2-3 days post infection and washed once in PBS prior to lysis and analysis of protein production as described in section 2.3

2.2.1.4 Identification and purification of recombinant baculovirus

The passage 4 virus stock contains the required recombinant virus mixed with non­ recombinant and single crossover viruses (O'Reilly, 1994) which affect the maximum levels of protein expression. If protein expression was low, recombinant viruses were plaque purified (O'Reilly, 1994) 149-165) to obtain clonal isolates of a potential recombinant virus, which was then used to make a high titre virus stock .

2.2.1.5 Protein expression in Sf9 cells

Sf9 cells were infected with different amounts of the final amplified virus and harvested at different times between days 1-3 post infection. The levels of protein expression were monitored by analysing the whole cell lysates (section 2.3.1) at each time point using SDS-PAGE (2.3.4) and coomassie blue staining. In this way, the time required for maximum expression of the protein was determined.