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The Application of the Gibson Assembly Method in the Production of Two pKLS3 Vector-Derived Infectious Clones of Foot-and-Mouth Disease Virus
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Document Title
The Application of the Gibson Assembly Method in the Production of Two pKLS3 Vector-Derived Infectious Clones of Foot-and-Mouth Disease Virus
Author
Semkum P., Thangthamniyom N., Chankeeree P., Keawborisuth C., Theerawatanasirikul S., Lekcharoensuk P.
Affiliations
Department of Microbiology and Immunology, Faculty of Veterinary Medicine, Kasetsart University, Bangkok, 10900, Thailand; Center for Advanced Studies in Agriculture and Food, KU Institute for Advanced Studies, Kasetsart University, Bangkok, 10900, Thailand; Virology and Cell Technology Research Team, National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Pathum Thani, 12120, Thailand; Department of Anatomy, Faculty of Veterinary Medicine, Kasetsart University, Bangkok, 10900, Thailand
Type
Article
Source Title
Vaccines
ISSN
2076393X
Year
2023
Volume
11
Issue
6
Open Access
All Open Access, Gold, Green
Publisher
MDPI
DOI
10.3390/vaccines11061111
Format
Abstract
The construction of a full-length infectious clone, essential for molecular virological study and vaccine development, is quite a challenge for viruses with long genomes or possessing complex nucleotide sequence structures. Herein, we have constructed infectious clones of foot-and-mouth disease virus (FMDV) types O and A by joining each viral coding region with our pKLS3 vector in a single isothermal reaction using Gibson Assembly (GA). pKLS3 is a 4.3-kb FMDV minigenome. To achieve optimal conditions for the DNA joining, each FMDV coding sequence was divided into two overlapping fragments of approximately 3.8 and 3.2 kb, respectively. Both DNA fragments contain the introduced linker sequences for assembly with the linearized pKLS3 vector. FMDV infectious clones were produced upon directly transfecting the GA reaction into baby hamster kidney-21 (BHK-21) cells. After passing in BHK-21 cells, both rescued FMDVs (rO189 and rNP05) demonstrated growth kinetics and antigenicity similar to their parental viruses. Thus far, this is the first report on GA-derived, full-length infectious FMDV cDNA clones. This simple DNA assembly method and the FMDV minigenome would facilitate the construction of FMDV infectious clones and enable genetic manipulation for FMDV research and custom-made FMDV vaccine production. ? 2023 by the authors.
Industrial Classification
Knowledge Taxonomy Level 1
Knowledge Taxonomy Level 2
Knowledge Taxonomy Level 3
License
CC BY
Rights
Authors
Publication Source
WOS