5.2.1.1 Generation of recombinant human PON2 vector.
The human PON2 transcript (isoform 1, NCBI accession NM_000305.2, Ser311 variant) was
codon optimized for expression in insect cells and synthesized by DNA2.0 (Menlo Park, California, US) (Figure 5.2). The transcript included a BamHI restriction enzyme site seven bp upstream from the PON2 translation start codon, and a NotI restriction site immediately following the translation stop codon. Following restriction digestion, the PON2 transcript was ligated into the multiple cloning site of the pFastBac1 vector (Life Technologies,
Carlsbad, California) using the BamHI and NotI restriction sites to generate the recombinant human PON2 vector pFastBac1.hPON2 (Figure 5.3).
Figure 5.2 Nucleotide and protein sequence of synthesised human PON2 transcript. Figure shows human PON2 transcript (isoform 1, NCBI accession NM_000305.2, with Ser311) (top line) and translated AA sequence (middle line), with codon optimization for insect cell protein synthesis by DNA2.0. The transcript includes a BamHI restriction site seven bp upstream from the PON2 translation start codon, and a NotI restriction site immediately downstream from the translation stop codon.
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Figure 5.3 Vector map of pFastBac.hPON2, a hPON2-containing plasmid vector. The synthesized hPON2 sequence was inserted into the MCS of pFastBac to give pFastBac-hPON2, a 5797 bp plasmid with SV40 promoter and ampicillin and gentamycin resistance cassettes.
The pFastBac1.hPON2 construct was transformed into One Shot® TOP10 Competent Cells (Life Technologies) following to the manufacturer’s recommended chemical transformation protocol. Briefly, 5 μL of construct DNA was added to one vial of OneShot® TOP10
Competent Cells. The mixture was incubated on ice for 30 minutes, heat-shocked for 45 seconds at 42°C without agitation, and immediately chilled on ice for a further two minutes. A 250 μL aliquot of S.O.C. medium was added and the mixture shaken at 150 RPM at 37°C for one hour.
Transformed cells (20 μL, 50 μL and 120 μL aliquots) were then spread-plated on LB agar plates supplemented with 100 μg/mL ampicillin. The plates were incubated at 37°C overnight. The following day, single colonies were isolated from the plates and used to inoculate fresh LB-ampicillin plates. Selected colonies were used to inoculate 2 mL LB medium and grown overnight at 37°C, with shaking at 220 RPM. DNA from overnight cultures was isolated using a plasmid mini-prep kit (Promega, Madison, WI) (Chapter 2.4.2) and subjected to PCR using hPON2 primers (sequences in table 2.3) and gel electrophoresis
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used to verify correct insertion. DNA and glycerol stocks of pFastBac1.hPON2 were stored at −80°C for future use.
5.2.1.2 Generation of recombinant hPON2 bacmid and isolation of recombinant hPON2 bacmid DNA
The hPON2 bacmid was generated using the Bac-to-Bac® Baculovirus expression system
(Life Technologies) according to the manufacturer’s protocols. The pFastBac1 vector’s expression cassette recombines with the parent bacmid in the MAX Efficiency® DH10Bac
cells to form an expression bacmid. Briefly, 5 µL of pFastBac1.hPON2 DNA (200 pg/µL in TE, pH 8.0) was added to 100 µL of MAX Efficiency® DH10Bac™ in a pre-chilled 15-mL
polypropylene tube. The cell-DNA mixture was incubated on ice for 30 mins, heat shocked for 45 seconds at 42°C, then immediately chilled on ice for a further two minutes. Finally 10- fold serial dilutions were prepared in S.O.C medium before being plated onto LB plates supplemented with 50 μg/mL kanamycin, 7 μg/mL gentamicin, 10 μg/mL tetracycline, 40 μg/mL X-gal, and 40 μg/mL IPTG. The plates were incubated for 48 hours at 37°C after which time several white colonies were picked and struck onto fresh selective medium to obtain single colonies. A single colony was used to inoculate LB containing 50 μg/mL kanamycin, 7 μg/mL gentamicin and 10 μg/mL tetracycline and grown overnight at 37°C with shaking at 220 RPM. Bacmid DNA was then isolated using plasmid mini-preps (Promega) (Section 2.4.2.3) and PCR and gel electrophoresis was used to verify the presence of recombinant hPON2 constructs using primers: pUC/M13 primers (Table 2.3) The DNA and glycerol stocks of pFastBac1.hPON2 bacmid were stored at −80°C for future use.
5.2.1.3 Generation of recombinant hPON2 baculovirus stocks
Baculovirus stocks were generated in Spodoptera frugiperda Sf9 cells (Life Technologies), which were transfected with recombinant bacmid DNA using the Cellfectin® reagent supplied with the Bac-to-Bac® Baculovirus expression system (Life Technologies) and
following the manufacturer’s instructions. Briefly, recombinant hPON2 bacmid DNA was diluted to 500 ng/μL in TE Buffer, pH 8.0. Stationary phase Sf9 cells (~1 107 cells/mL)
were diluted to 2 106 cells/mL. In six-well plates, 2 mL of unsupplemented Grace’s Insect
Medium was added to each well followed by the addition 8 105 Sf9 cells in SF900III
medium. The cells were allowed to rest and attach to plates at room temperature for 15 minutes. The DNA–transfection reagent complexes were prepared at various ratios of DNA to transfection reagent in unsupplemented Grace’s Insect Medium to determine the
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added drop-wise to the cells. The cells were then incubated in a humidified incubator at 27°C for 6 hours before growth medium was replaced with SF900-III serum-free medium and further incubated in a humidified incubator at 27°C. Cells were monitored daily for signs of viral infection (Table 5.1). Once cells showed signs of late infection (Table 5.1), the growth medium from the wells was removed and centrifuged at 500 g for five minutes to remove any cells and debris. The supernatant containing virus particle was collected and FBS was added to a final concentration of 2% (v/v). This yielded the P1 baculoviral stock. P1 baculoviral stock was stored short-term at 4°C, protected from light, or at −80°C for long- term storage. Immunoblot analysis of cell lysates was carried out with PON2 antibody to establish the presence of rhPON2 protein and viral titre determined by the endpoint titre method as described in Chapter 2 (Section 2.6.4). For long-term storage, the P1 baculoviral stock was stored at −80°C.
The baculoviral stock was further amplified by infecting more Sf9 cells. Stationary phase Sf9 cells were diluted to 2 106 cells/mL in 50mL in a vented, flat-bottom polycarbonate
Erlenmeyer flask. P1 baculovirus was added to the cells at a multiplicity of infection (MOI) of 0.1. The inoculum required was calculated using the following formula:
Inoculum required (mL) = MOI ( pfu
cell) × number of cells baculovirus stock titer (pfumL)
The inoculated Sf9 cells were incubated at 27°C, with shaking at 120 RPM in the dark. After 48 hours, the cell suspension was collected and centrifuged at 500 g for five minutes to remove any cells and debris. The supernatant was collected and FBS added to a final concentration of 2% (v/v), which constituted the P2 baculoviral stock. P2 baculoviral stock
Table 5.1 Characteristics of viral infection in Sf9 and High Five™ insect cells.
Adapted from Bac-to-Bac baculovirus expression system user guide, 2013. [275]
Signs of Infection Phenotype Description
Early
(First 24 hours)
Increased cell diameter A 25%–50% increase in cell diameter may be seen.
Increase in cell nuclei size Nuclei may appear to "fill" the cells. Late
(24–72 hours)
Cessation of cell growth Cells appear to stop growing when compared to a cell-only control.
Granular appearance Signs of viral budding; vesicular
appearance of cells.
Detachment Cells release from the plate or flask.
Very late (>72 hours)
Cell lysis Cells appear lysed, and show signs of
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was stored as described for P1 stocks and viral titre was determined by the endpoint titre method.