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Design and application of expression constructs for FMDV serotype O structural proteins
Mostafa R Zaher1,2, Dalia M El-Husseini3, Mohamed H El-Husseiny4
1Genome Research Unit, Animal Health Research Institute, Agriculture Research Center (ARC), Giza, 12618, Egypt. mostafa.r.zaher@ahri.gov.eg.
None:
Design and validate flexible constructs for recombinant expression of FMDV serotype O structural proteins of the circulating topotypes using newly designed degenerate primers. The designed degenerate primers targeting diverse topotypes enabled the successful amplification of VP0, VP1, and VP3 genes. Integration of the essential transcriptional and translational regulatory elements including T7 promoter, leader g10 sequence, and T7 terminator, as well as ribosome binding site (RBS), start and stop codons, respectively via overlap extension PCR empowered efficient expression of these proteins in E. coli. Cloned constructs expressed the target proteins of expected molecular weights: VP0 (34 kDa), VP1 (24 kDa), and VP3 (22 kDa). SDS-PAGE and Western blotting confirmed high protein yield and purity. This platform demonstrated adaptability for diagnostic and vaccine development applications. The workflow offers a robust tool for producing FMDV structural proteins concerning the circulating strains attempting to improve control measures including diagnosis and vaccinations.
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