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again using DNA derived from cells at passage 9, this time to confirm integration of the entire gene cassette into the MVA genome.
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the cells lifting. On day 4 the cells were harvested, and DNA was extracted using the aforementioned QIAGEN® DNeasy Blood and Tissue Kit. PCR was conducted as outlined in Table 2.1 using the primers reflected in Table 2.2. The reaction was performed using the KAPA 2G Robust HotStart ReadyMix kit in accordance with the protocol provided. PCR reactions were made up to a total of 50 µl, with 5 µl of DNA in each reaction. An aliquot of 25 ul KAPA2G Robust HotStart ReadyMix (Sigma-Aldrich, South Africa) containing dNTPs, a KAPA2G Robust DNA polymerase and a buffer, was added. The volume was adjusted with sterile dH20 to make a total volume of 50 µl. The reactions were set up for 35 cycles, with cells only (no DNA) serving as a negative control; and parent MVA serving as a positive control. A 20 µl aliquot of the PCR products was resolved on a 0.6% agarose gel by electrophoresis as outlined in Section 2.1.2. The PCR binding sites and expected fragment sizes generated by the different primer pairs are depicted in Figure 2.3.
2.5.2 DNA sequencing
The PCR products were sequenced using the Sanger method by the Central Analytical Facilities at the University of Stellenbosch (South Africa). This was done to confirm the genetic integrity of the inserted gene, SΔTM using the sequencing primers outlined below in Table 2.3. PCR products were amplified using primers PEPV DRfwd and G1Lr2 and 100 ng/µl with 10 µl sample per primer pair was sent to be sequenced. Sequencing primers were designed to bind to the SΔTM gene insert.
The sequence was analysed using CLC Genomics Workbench which enabled the sequence reads to be aligned to the reference sequence.
Table 2.3: Primers used to sequence MVA-SARS-2-SΔTM
Primer Sequence
PEPV DRfwd 5’- GGGTGTAGCTAACAACCCTG - 3'
FNK1 5’- GTGAACCTGACTACCAGAAC – 3'
FNK2 5’ - GGATGGAGAGCGAGTTTAGGG – 3'
FNK3 5’ - GGATGGAGAGCGAGTTTAGGG – 3'
FNK4 5’ - GGTGCTGAGCTTTGAGCTGC – 3'
FNK5 5’ - CGCCTATACCATGTCCCTGG – 3'
FNK6 5’ - GATGATCGCCCAGTACACATC – 3'
FNK7 5’ - GCTATCACCTGATGAGCTTC – 3'
FNK8 5’ - GCCTACGTGAGAAAGGATGG – 3'
FNK9 5’ - TTT TCT GTC GAC GGG CCC G – 3'
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Table 2.1: PCR cycling parameters for amplifying MVA-SARS2-S∆TM genomic DNA
Reaction Stage Temperature (˚C) Duration
Initial denaturation 95 3 minutes
Denaturation 95 15 seconds
Annealing 53 15 seconds
Extension 72 6 minutes
Final extension 72 7 minutes
Table 2.2: Primers used for PCR validation of MVA-SARS2-S∆TM
Set Primer Primer sequence Binding site
1 (Left end rMVA)
I8Rf2 5’ - AGATGAACTTCAGGGTCAGC – 3' Outside I8R flank eGFPrev 5’ - GAAAAGGAAGAGTAGGAAGAG –
3'
eGFP gene 2 (Right end
rMVA)
PEPVDRfwd 5’ - GGGTGTAGCTAACAACCCTG – 3' Outside K1L gene G1Lr2 5’ - GGCCATGTGTAACACTAGAG – 3' Outside G1L flank 3 (wtMVA) I8Rf2 5’ - GAAAAGGAAGAGTAGGAAGAG –
3'
Outside I8R flank
G1Lf2 5’ - GGCCATGTGTAACACTAGAG – 3' Outside G1L flank
Figure 2.3: Schematic diagrams showing the binding sites of primer set 1 (I8Rf2 and eGFPrev) and primer set 2 (PEPV DRfwd and G1Lr2) in the recombinant virus (top) and primer set 3 for the wildtype parent MVA (bottom). PCR products are shown with expected product sizes as horizontal lines.
64 2.5.3 Western blot analysis
2.5.3.1 Preparation of media and cell lysate
Following transfection or infection, the spent growth media was removed and stored in Eppendorf tubes. The cells were rinsed with 500 µl 1XPBS. An aliquot of 200 µl Glo Lysis Buffer (Promega, South Africa) was dispensed over the cells before swirling the plate to ensure full coverage with the buffer. The cells were left at room temperature for 5 minutes to allow lysis to occur. The lysed cells were centrifuged for 10 minutes at 2500 x g to pellet insoluble host cell debris, and the supernatant was retained at –20 ˚C.
2.5.3.2 Sodium dodecyl sulphate - polyacrylamide gel electrophoresis (SDS PAGE)
Aliquots of 30 µl of media or lysate were mixed with 10 µl aliquots of 4X protein loading dye (Appendix A) and boiled at 95 ˚C for 5 minutes to denature the protein. Samples were loaded (15 – 20 µl) into the wells of 8% SDS page gels, alongside 10 µl Colour Protein Standard Broad Range (New England BioLabs, UK). The samples were resolved for 2 hours at 200 V in 1 X running buffer (Appendix A).
2.5.3.3 Immunoblotting of protein
The protein was transferred from the SDS gel to an Immuno-Blot PVDF membrane (Bio-Rad, USA) using the Bio-Rad Trans-Blot SD Semi-Dry Electrophoretic Transfer Cell apparatus (Bio- Rad, South Africa). A PVDF membrane (Bio-Rad, South Africa) was pre-soaked in methanol (Sigma-Aldrich, France) for 30 seconds to activate the membrane for protein binding. The membrane and extra thick blotting paper were both soaked in transfer buffer (Appendix A) for 5- 10 minutes. The SDS-PAGE gel was placed on top of the activated PVDF membrane and the gel/membrane complex was sandwiched between the extra thick blotting paper. Transfer was conducted for 60 minutes at 25 V.
Following transfer, the membranes were blocked with 5% skim milk in PBS (Lonza, Belgium) for 2 hours at room temperature, whilst gently shaking. The blocked membranes were then incubated with a 1:5000 dilution of rabbit anti-SARS-CoV-2 Spike Glycoprotein S1 antibody (ab281311) (Abcam, South Africa) overnight. Membranes were washed with PBST wash buffer (Appendix A)
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four times for 15 minutes, each with gentle agitation. The buffer was discarded, and the secondary goat anti-rabbit IgG (whole molecule) alkaline phosphate antibody (A3687) (Sigma-Aldrich, USA) (diluted 1:5000) was added to the membrane. The membrane was incubated at room temperature for 2 hours whilst shaking. The PBST washing steps were repeated as before, and the Western blot was developed for up to 1 hour using NBT/BCIP solution (KPL/SeraCare, USA).
The reaction was terminated by washing the membrane in RO water.
2.5.4 Immunostaining of protein
A 12-well plate was seeded with 5 x 105 cells/ml in each well and then infected with MVA-SARS2- SΔTM. The infected cells were incubated for 72 hours. The spent media was removed and the cells were fixed with 500 µl 4% paraformaldehyde (Appendix A) with gentle agitation for 10 minutes.
A 500 µl aliquot of 100% methanol (Sigma-Aldrich, France) was added and the plate was agitated for 1 minute. Cells were washed with PBS to remove excess methanol, before being blocked with 2% BSA (Appendix A) for 1 hour at room temperature. An aliquot of 500 µl SARS-CoV-2 spike rabbit primary antibody (1:500) was added to each well and the plate was incubated at room temperature for 90 minutes. Cells were again washed with PBS before an aliquot of 500 µl donkey anti-rabbit Cy3 conjugated secondary antibody (1:500) was added to each well and the incubation was repeated as before. The cells were washed twice with PBS, viewed using a Zeiss Axio Vert.A1 fluorescent microscope, and then imaged.