Random mutagenesis of BoNT/E Hc nanobody to construct a secondary phage-display library.
OBJECTIVE
To construct secondary mutant phage-display library of recombinant single variable domain (VHH) against botulinum neurotoxin E by error-prone PCR.


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.


CONCLUSIONS
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.
Department of Biology, Basic Science Faculty, Shahed University, Tehran, Iran.
B
B
Shahi
S L
SL
Mousavi Gargari
I
I
Rasooli
M
M
Rajabi Bazl
R
R
Hoseinpoor
D000818:Animals; D000907:Antibodies, Bacterial / Q000737:chemistry / Q000235:genetics*; D000915:Antibody Affinity; D000918:Antibody Specificity; D001435:Bacteriophages / Q000235:genetics; D001905:Botulinum Toxins / Q000276:immunology*; D002162:Camelus / Q000276:immunology; D061505:Cell Surface Display Techniques; D015723:Gene Library; D016296:Mutagenesis; D016133:Polymerase Chain Reaction; D061905:Single-Domain Antibodies / Q000737:chemistry / Q000235:genetics*
D000907:Antibodies, Bacterial; D061905:Single-Domain Antibodies; D001905:Botulinum Toxins; C096326:botulinum toxin type E
BoNT/E; affinity maturation; error-prone PCR; nanobody; phage-display library