Random mutagenesis of BoNT/E Hc nanobody to construct a secondary phage-display library.

AIMS: To construct secondary mutant phage-display library of recombinant single variable domain (VHH) against botulinum neurotoxin E by error-prone PCR. METHODS AND RESULTS: The gene coding for specific VHH derived from the camel immunized with binding domain of botulinum neurotoxin E (BoNT/E) was amplified by error-prone PCR. Several biopanning rounds were used to screen the phage-displaying BoNT/E Hc nanobodies. The final nanobody, SHMR4, with increased affinity recognized BoNT/E toxin with no cross-reactivity with other antigens especially with related BoNT toxins. CONCLUSIONS: The constructed nanobody could be a suitable candidate for VHH-based biosensor production to detect the Clostridium botulinum type E. SIGNIFICANCE AND IMPACT OF THE STUDY: Diagnosis and treatment of botulinum neurotoxins are important. Generation of high-affinity antibodies based on the construction of secondary libraries using affinity maturation step leads to the development of reagents for precise diagnosis and therapy.