Generation and molecular docking analysis of specific single-chain variable fragments selected by phage display against the recombinant nucleocapsid protein of fig mosaic virus
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Published on: July 15, 2019
Morteza Shahmirzaie1, Mohammad Reza Safarnejad2, Farshad Rakhshandehroo3
1Department of Plant Protection, College of Agricultural Sciences and Food Industries, Science and Research Branch, Islamic Azad University, Tehran, Iran; Pharmaceutical Sciences Research Center, Shahid Beheshti University of Medical Sciences, Niayesh Highway, Valiasr Ave, Tehran, Iran.
Abstract:
The mosaic disease caused by fig mosaic virus (FMV) is considered the plague of fig worldwide. A naïve phage display library, raised against the recombinant nucleocapsid protein of FMV (FMV-Np) was screened to obtain specific monoclonal recombinant antibodies in the form of single chain variable fragments (scFvs). After three rounds of biopanning, the bacterially expressed FMV-Np was used as an antigen for selecting specific phages for the production of specific soluble scFvs to be used in immunological assays. The binding specificity of scFvs against FMV-infected fig samples was evaluated by immunoblotting and Plate trapped antigen-ELISA (PTA-ELISA), which revealed efficient of the resultant scFvs to the target antigen. Silico homology-modelling and molecular docking analysis confirmed the scFv and FMV-Np interactions with the anti-FMV-Np scFv through an estimated binding energy of -650 kj mol-1; considered to be generated from the interactions between 13 amino acids residues predicted as putative epitopes in the interface pocket of FMV-Np and scFv antibody. This high affinity was further confirmed in the specificity of ELISA and immunoblotting assays. This is the first report on the application of phage display technology to generate specific recombinant scFvs against FMV that can be applied in development of antibody-mediated protection strategy to control the fig mosaic disease.
Insights
Researchers developed specific single chain variable fragments (scFvs) against fig mosaic virus (FMV) using phage display technology. These scFvs show high affinity and specificity, offering a new tool for controlling FMV disease in figs.
Area of Science:
- Plant Virology
- Molecular Biology
- Immunotechnology
Background:
- Fig mosaic virus (FMV) causes a devastating mosaic disease in figs globally.
- Effective diagnostic tools and control strategies for FMV are crucial for fig cultivation.
Purpose of the Study:
- To generate specific monoclonal recombinant antibodies against FMV nucleocapsid protein (FMV-Np).
- To evaluate the utility of these antibodies in immunological assays for FMV detection.
- To explore phage display technology for developing novel tools against FMV.
Main Methods:
- Phage display technology was employed to screen a library against recombinant FMV-Np.
- Biopanning and bacterial expression were used to select and produce specific single chain variable fragments (scFvs).
- Immunoblotting and Plate trapped antigen-ELISA (PTA-ELISA) were utilized to assess scFv binding specificity.
- In silico homology modeling and molecular docking were performed to analyze antibody-antigen interactions.
Main Results:
- Specific scFvs with high binding affinity and specificity to FMV-Np were successfully generated.
- The developed scFvs demonstrated efficient binding to FMV-infected fig samples in immunological assays.
- Molecular modeling predicted strong interactions between scFvs and FMV-Np, with an estimated binding energy of -650 kJ mol⁻¹.
- The study identified 13 amino acid residues involved in the putative epitope-paratope interactions.
Conclusions:
- Phage display technology is effective for generating specific recombinant scFvs against FMV.
- The characterized scFvs are suitable for immunological assays and can aid in FMV diagnosis.
- These findings pave the way for developing antibody-mediated strategies to control fig mosaic disease.
