Cloning, Expression and Functionality evaluation of Recombinant Monoclonal antibody against VP1 capsid protein of FMD virus

Articles in Press

Document Type : Original Articles

Authors

1 Department of Pathobiology, Science and Research Branch, Islamic Azad University, Tehran, Iran.

2 Razi Vaccine and Serum Research Institute (RVSRI), Agricultural Research, Education and Extension Organization (AREEO), Karaj, Iran.

3 Department of Microbiology, Karaj Branch, Islamic Azad University, Karaj, Iran.

10.22092/ari.2025.368226.3491

Abstract

Foot-and-Mouth Disease (FMD) is one of the most contagious viral disease with potentially devastating economic, social and environmental impacts that caused by a virus of the genus Aphthovirus, family Picornaviridae. FMD virus (FMDV) is a highly variable RNA virus, and there is little or no cross-protection between serotypes and even between different strains of the same serotype.
Monoclonal antibody (Mab) has a pivotal role in detection and serotyping of FMDV in pathological specimens, and also protection evaluation against FMD after vaccination. This study explore the expression and function of an engineered recombinant single-chain variable fragment (ScFv-Mab) in Escherichia coli (E.coli) BL21 (DE3) rosetta strain. Production of recombinant Mab against FMDV in prokaryotic system is a cheap, cost effective, simple and high yield that can reduce production complexities.
Designed ScFv-Mab gene ordered into pET28a (+) expression plasmid. Expressed protein purified using Ni2+-NTA resin column and quality assessed by 12% SDS-PAGE. Finally, efficiency and functionality of a ScFv- Mab confirmed by indirect sandwich (capture) ELISA.
The transformed E. coli BL21 (DE3) rosetta strain induced with 0.5 mM IPTG and incubated for 12 hr at 37 ˚C. Significant protein expressed in both soluble and insoluble fractions with purity of > 90%. The concentration of purified ScFv-Mab in optimum condition calculated approximately 2.00 mg/ml by Bradford assay. By analyzing mean of negative serum and checkerboard in 1:10 dilution, 400 ngr of ScFv-Mab coated in each well. Eventually optical density (OD) of 0.3 selected as cut-off in the indirect Sandwich ELISA assay. The ELISA results showed that the ScFv-Mab fragment successfully detected serotype O of FMDV with signals of 0.3 ≥ X ≤ 1. 5 profiles at 450nm wavelength in different positive control treatments.

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Articles in Press, Corrected Proof
Available Online from 02 February 2025

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