ADENOVIRUS GENOME CONSTRUCTION WITH LAC OPERON COMPONENT FOR COVID-19 VACCINE CANDIDATE PRODUCTION
The development of adenovirus based COVID-19 vaccine used as delivery vector for SARS-CoV-2 virus spike gene is carried out to handle pandemic. Lac System is a transcriptional regulatory system that can be used to inhibit Spike protein expression in host cells for optimal adenovirus production...
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| Format: | Theses |
| Language: | Indonesia |
| Online Access: | https://digilib.itb.ac.id/gdl/view/76601 |
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| Institution: | Institut Teknologi Bandung |
| Language: | Indonesia |
| Summary: | The development of adenovirus based COVID-19 vaccine used as delivery vector
for SARS-CoV-2 virus spike gene is carried out to handle pandemic. Lac System is
a transcriptional regulatory system that can be used to inhibit Spike protein
expression in host cells for optimal adenovirus production. This study aims to
construct an adenovirus genome with the lac operon as a regulatory component
of Lac System for the production of COVID-19 vaccine candidate. Synthetic gene
was designed having components of the lac operon, SV40 intron, and multiple
cloning sites (MCS). Restriction enzyme sites were mapped on the synthetic gene
design for pShuttleCMV-lacO-Spike and pShuttleCMV-lacO-Intron-Spike
plasmids construction using restriction and ligation methods. Homologous
recombination between pShuttleCMV-lacO-Spike and pAdEasy adenovirus
genome was successfully performed in Escherichia coli BJ5183. The presence of
hexon gene, spike gene, and lac operon on pAdEasy-lacO-Spike was identified
using PCR with specific primers. The results of PCR analysis showed
experimental size of 146 bp for hexon gene (theoretical size 168 bp), 3,872 bp for
spike gene (theoretical size 3,848 bp), and 2,201 bp for lac operon (theoretical
size 2,070 bp), proving the existence of the three genes on pAdEasy-lacO-Spike.
Initial adenovirus production was performed by pAdEasy-lacO-Spike transfection
in AD293 cells, using pAdEasy-Spike and pAdyEasy-lacZ as controls. Adenovirus
production success was confirmed using PCR with specific primers. The results of
PCR analysis showed experimental size of 148 bp for the hexon gene (theoretical
size 168 bp) and 183 bp for the spike gene (theoretical size 183 bp). Adenovirus
titers produced from transfection were 6.12 × 105
IFU/mL for AdV-lacO-Spike,
7.65 × 105
IFU/mL for AdV-Spike, and 1.07 × 106
IFU/mL for AdV-lacZ.
Adenovirus titers of AdV-lacO-Spike was comparable to AdV-Spike indicated
initial production success of AdV-lacO-Spike, with pAdEasy-lacO-Spike
adenovirus production capability comparable to pAdEasy-Spike.
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