Construction, expression and functional analysis of anti-clenbuterol codon-optimized scFv recombinant antibody

Area of Science:

• Biotechnology
• Molecular Biology
• Immunochemistry

Background:

• Clenbuterol (CBL) is a banned substance in livestock due to its potential health risks.
• Sensitive and reliable detection methods for CBL are crucial for food safety and regulatory compliance.
• Recombinant antibody fragments, such as single-chain variable fragments (scFv), offer advantages in developing rapid detection tools.

Purpose of the Study:

• To construct and express a codon-optimized single-chain variable fragment (coscFv) against clenbuterol (CBL) using an Escherichia coli system.
• To optimize the expression and purification of coscFv.
• To functionally characterize the coscFv for its ability to detect CBL.

Main Methods:

• Codon optimization for coscFv expression in E. coli.
• Optimization of ionic concentration for coscFv expression.
• Optimization of inclusion body extraction and affinity purification of coscFv.
• Indirect competitive enzyme-linked immunosorbent assay (icELISA) for functional analysis.
• Molecular docking to determine binding interactions.

Main Results:

• Optimized ionic concentration increased coscFv yield to 23.26%.
• Purified coscFv exhibited a molecular weight of approximately 27 kDa.
• CoscFv constituted 43.9% after optimized extraction and purification.
• The coscFv demonstrated high sensitivity for CBL detection with an IC50 of 4.22 ng/mL.
• Molecular docking identified key amino acid residues involved in CBL binding.

Conclusions:

• The codon-optimized single-chain variable fragment (coscFv) was successfully constructed and expressed in E. coli.
• Optimized expression and purification protocols significantly improved coscFv yield and purity.
• The developed coscFv specifically recognizes and binds to clenbuterol (CBL).
• The coscFv is a promising candidate for the sensitive and specific detection of clenbuterol in various applications.