Deletion Mutant Comprising 198 Residues of BoNT/A Toxin Receptor Binding Domain Retained GT1b Binding Property but Failed to Induce Protective Antibody Response in a Mouse Model
2012
The most effective protection against toxin is inducing protective immune response through vaccination that
will produce neutralizing antibodies. Antibodies will bind to and clear toxin from the circulation before it can enter nerve
cells and block neurotransmission and can also be used for development of detection system. In the present study we constructed
a deletion mutant of the binding domain (1098-1296) to produce smallest toxin fragment as vaccine candidate
against BoNT/A. The BoNT/A-HCC protein was highly expressed in Escherichia coli SG13009 and found to form inclusion
bodies. The purified inclusion bodies were solubilized in 6 M guanidine-HCl containing 10 mM β-mercaptoethanol
and 20 mM imidazole and the rBoNT/A-HCC was purified and refolded in a single step on Ni2+ affinity column. The purified
protein was ~98 % pure as assessed by SDS–polyacrylamide gel with the yield of 8 mg/L and showed binding to
polysialoganglioside (GT1b). The rBoNT/A-HCC at dose of 40 μg/mouse generated high IgG antibody titre with predominance
of IgG1 subtype, but failed to protect animals against BoNT/A challenge. Antibody titre in serum was determined
by enzyme linked immunosorbent assay and specific binding to rBoNT/A-HCC was demonstrated by surface plasmon
resonance (SPR), with a dissociation constant of 0.8 pM.
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