Random mutagenesis of BoNT/E Hc nanobody to construct a secondary phage-display library
Article first published online: 16 MAY 2014
© 2014 The Society for Applied Microbiology
Journal of Applied Microbiology
Volume 117, Issue 2, pages 528–536, August 2014
How to Cite
Shahi, B., Mousavi Gargari, S.L., Rasooli, I., Rajabi Bazl, M. and Hoseinpoor, R. (2014), Random mutagenesis of BoNT/E Hc nanobody to construct a secondary phage-display library. Journal of Applied Microbiology, 117: 528–536. doi: 10.1111/jam.12526
- Issue published online: 18 JUL 2014
- Article first published online: 16 MAY 2014
- Accepted manuscript online: 25 APR 2014 08:22AM EST
- Manuscript Accepted: 4 APR 2014
- Manuscript Revised: 23 MAR 2014
- Manuscript Received: 7 FEB 2014
- Shahed University
- affinity maturation;
- error-prone PCR;
- phage-display library
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.
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.