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Volume 21, Issue 17
  • ISSN: 1570-1808
  • E-ISSN: 1875-628X

Abstract

Background

The adenovirus vector has been widely studied for vaccines and gene therapies. During the production of the adenovirus vector, a high virus titer is desired to obtain enough virus. The adenovirus vector has been widely studied for the vaccinations and gene therapies, where a high virus titer is desired to obtain sufficient quantities of the virus. For an adenovirus vector-based vaccine, suppression of antigen expression during production would improve the virus titer during production.

Objective

This study aimed to construct an adenovirus vector with lacO-regulated antigen expression using the SARS-CoV-2 spike as a transgene model, which would improve the adenovirus titer during production.

Methods

The lacO expression cassette was designed and prepared as a synthetic gene in pUC57. The lacO expression cassette was then subcloned into pShuttle-CMV. The SARS-CoV-2 spike gene was then inserted into the pShuttle-CMV harboring lacO to generate pShuttle-lacO_S and pShuttle-lacO-intron_S. Recombinant pShuttle was then used to generate a recombinant adenovirus genome using BJ5183 pAdeasy-1. Transfection of the I-linearized adenovirus genome into AD293 and HEK293 cells was used to generate adenovirus primary stock for 14 days of incubation.

Results

Recombinant adenovirus genomes, pAdeasy-lacO_S and pAdeasy-lacO-intron_S, were successfully generated and characterized using I restriction and PCR. In the production of adenovirus primary stocks, the adenovirus titer produced in AD293 cells was higher than in HEK293 cells. The primary stock titer of AdV_lacO-intron-S was higher than AdV_lacO-S and AdV_S titers.

Conclusion

Production of adenovirus with lacO and spike gene, either with or without intron, was successful with a higher titer as compared to AdV_S titer.

© 2024 Bentham Science Publishers
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2024-05-21
2025-07-07

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Construction of an Adenovirus Vector with Laco-regulated Antigen Expression Using the SARS-Cov-2 Spike as a Transgene Model
Letters in Drug Design & Discovery 21, 3723 (2024); https://doi.org/10.2174/0115701808293712240516105131
/content/journals/lddd/10.2174/0115701808293712240516105131
/content/journals/lddd/10.2174/0115701808293712240516105131
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  • Article Type:     Research Article
Keyword(s): Adenovirus; COVID-19; intron; LacO; primary stock; spike

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