3.1 Construction of a mammalian expression plasmid encoding SARS-CoV-2 S∆TM
3.2.1 Construction of transfer vector pMVA-FNK1
The backbone of the transfer vector encoding SARS-CoV-2-SΔTM was derived from pSSPEx:
Du151 Gly V2.1, as described by Van Diepen and colleagues [235]. The pSSPEx: Du151 Gly V2.1 plasmid was developed in a separate study and instead of the CAP256 had the Du151 gene.
pSSPEx: Du151 Gly V2.1 contains the gene for a synthetic Du151 HIV-1 Envelope gp150 gene under the control of the mH5 promoter, a K1L selection gene under the control of the vaccinia virus pSS promoter and an eGFP gene under control of the vaccinia virus p7.5 promoter. These genes are placed between sequences homologous to the 3’ ends of the convergent MVA ORFs I8R and G1L. K1L is a host range gene from vaccinia virus which allows for viral growth in the rabbit kidney cell line, RK13. RK13 cells, are non-permissive to the wildtype MVA virus, but permissive to the virus expressing K1L. This means that only the recombinant MVA can grow in RK13 cells thereby enabling its purification from unwanted parental virus. Additionally, eGFP is present as a fluorescent marker to enable the growth of the recombinant virus to be monitored. In order to insert the SARS-CoV-2 SΔTM gene into MVA, the HIV Env gene in the pSSPEx Du151 Gly V2.1 plasmid vector backbone was replaced with the SARS-CoV-2 SΔTM gene. Figure 3.7 shows the cloning strategy employed to modify the transfer vector. The resulting plasmid has the SARS- CoV-2 SΔTM gene downstream of the vaccinia virus mH5 promoter.
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Figure 3.7: Overview of the cloning strategy used for the construction of transfer vector pMVA-FNK1. The pUC57:SARS-CoV-2-SΔTM and pSSPEx Du151 Gly V2.1 plasmids were both digested with EcoRI and HindIII to release the SΔTM gene and pSSPEx vector backbone respectively. The gene was then ligated into the vector backbone to form the plasmid pMVA-FNK2. (ColE1 Origin = E.coli origin of replication, CmpR = Chloramphenicol resistance gene, G1L = MVA right flank, I8R = MVA left flank; eGFP = green fluorescent protein, K1L = host range gene, p7.5
= early/late vaccinia virus promotor, pSS = synthetic vaccinia virus promotor, mH5 = vaccinia virus modified H5 promotor).
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Restriction enzymes EcoRI and HindIII were used to digest the pSSPEx Du151 Gly V2.1 plasmid thus freeing the backbone. This in turn enabled the insertion of the SARS-CoV-2-SΔTM gene, which was excised from pUC57 using the same enzymes, EcoRI and HindIII. This approach generated complementary sticky between the vector backbone and the antigen gene sequence. As described in Section 2.1.3, restriction products were resolved on a 0.8% agarose gel and fragments corresponding to the pSSPEx Du151 Gly V2.1 backbone (5.5 kb) and the SARS-CoV-2-SΔTM gene (3.8 kb) were recovered from the gel (Figure 3.8A). The gene was ligated into the pSSPEx Du151 Gly V2.1 in place of the HIV Env gene, thus forming the transfer vector pMVA-FNK1.
E.coli cells were transformed with the ligated products. Putative clones were then grown in LB media and subjected to small-scale DNA isolation. The resulting DNA samples were screened with restriction endonucleases EcoRI and HindIII to identify clones of the desired recombinant plasmid and the resulting restriction fragments were resolved by agarose gel electrophoresis (Figure 3.8B).
The desired clones were expected to yield products of sizes 3.8 kb and 5.5 kbwhich correspond to the SΔTM insert and vector backbone, respectively.
Figure 3.8: Construction of transfer vector pMVA-FNK1 containing the SARS-CoV-2 SΔTM gene sequence and screening for putative clones. A) Recovery of the pSSPEx backbone and SARS-CoV-2-SΔTM gene following restriction digestion and resolution of the resulting DNA products by agarose gel electrophoresis. 1) pSSPEx Du151 Gly V2.1 backbone; 2) pUC57:SARS-CoV-2-SΔTM. Both plasmids were digested with restriction endonucleases EcoRI and HindIII to free the pSSPEx vector backbone and the S∆TM gene respectively. B) Screening of putative pMVA-FNK1 clones. DNA was digested with restriction endonucleases EcoRI and HindIII. Samples were subjected to electrophoresis on a 0.8% agarose gel at 100 V for 45minutes alongside a 5µl aliquot of GeneRuler 1 kb DNA Ladder (MW).
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One of the putative clones (lane 3) was grown up in 100 ml LB media and the culture was subjected to a large-scale DNA isolation to recover plasmid DNA for transfections. The integrity of the plasmid was also verified by restriction enzyme mapping as shown in Table 3.2 and Figure 3.9.
The clone was mapped using restriction endonucleases. This yielded fragments of the expected sizes indicating that the correct eluted fragment was cloned. EcoRI and HindIII (Figure 3.8, lane 2) produced 3 fragments of 3842, 5441 and 9282 bp instead of 2 fragments of 3842 and 5441 bp.
This could be explained by partial digestion of the full linear fragment.
BamHI and NcoI produced fragments of the anticipated size, indicating that the plasmid was indeed correct.
Table 3.2: Restriction endonucleases used to confirm the integrity of the plasmid pMVA-FNK1.
Figure 3.9: Confirmation of the integrity of the plasmid, pMVA-FNK1. The plasmid was digested with restriction endonucleases. 1) No endonuclease control; 2) EcoRI and HindIII; 3) BamHI; 4) NcoI.
Samples were analysed by 0.8% agarose gel electrophoresis alongside an aliquot of 5 µl GeneRuler 1 kb DNA Ladder (MW)
Restriction enzyme
(s) Expected products (bp)
EcoRI - HindIII 3842 bp, 5441 bp
BamHI 3731 bp, 5552 bp
NcoI 1808 bp, 1838 bp, 5577 bp
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