Cloning, Expression and Functionality Evaluation of Recombinant Monoclonal Antibody Against VP۱ Capsid Protein of FMD Virus

Introduction: Foot-and-mouth disease (FMD) is a highly contagious viral disease caused by the FMD virus (FMDV), which belongs to the Aphthovirus genus in the Picornaviridae family. FMDV is a highly variable RNA virus, and there is limited cross-protection between serotypes and strains. The disease can have devastating economic, social, and environmental impacts. The FMDV capsid is composed of VP۱-VP۴ structural proteins, with VP۱ being the most abundant protein consisting of ۲۱۳ amino acids. The G-H loop (residues ۱۴۱-۱۶۰) of the VP۱ capsid protein is highly variable and serves as the main antigenic region. It contains a conserved triplet of amino acids, Arg-Gly-Asp (RGD), which induces the production of protective antibodies against various FMDV types. Monoclonal antibodies (mAbs) play a crucial role in detecting and serotyping FMDV in pathological samples, as well as evaluating protection post-vaccination against FMD.Materials & Methods: This study explores the expression and function of an engineered recombinant single-chain variable fragment (scFv-mAb) in Escherichia coli BL۲۱ (DE۳) Rosetta strain as a cost-effective prokaryotic system with high yield. The scFv-mAb gene was inserted into the pET۲۸a (+) expression plasmid. E. coli cells were transformed with the plasmid, induced with ۰.۵ mM IPTG, and incubated at ۳۷ ˚C for ۱۲ hours. The protein was purified using a Ni۲+-NTA resin column and analyzed by ۱۲% SDS-PAGE for quality assessment. The efficiency and functionality of the scFv-mAb were confirmed using an indirect sandwich (capture) enzyme-linked immunosorbent assay (ELISA).Results: The purified scFv-mAb concentration was determined to be approximately ۲.۰۰ mg/mL using the Bradford assay under optimal conditions. Each well was coated with ۴۰۰ ng of scFv-mAb based on checkerboard results and the mean of negative serum at a ۱:۱۰ dilution. The indirect sandwich ELISA assay yielded an optical density (OD) signal range of ۰.۳ to ۱.۵ at a ۴۵۰ nm wavelength in different positive control treatments.Conclusion: The ELISA results showed that the scFv-mAb fragment successfully detected serotype O of FMDV. Further research could confirm the potential of this recombinant antibody for broader commercial applications in the future.

کلیدواژه ها:

Foot-and-mouth disease (FMD) ، G- H loop ، VP۱ ، Recombinant ScFv-Mab ، Indirect sandwich enzyme-linked immunosorbent assay (ELISA)