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

Title: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

Volume: 19 Issue: 5

Author(s): Manglesh Kumar Singh, Garima Gupta, Mannan Boopathi, Pallavi Gupta, Vinita Chauhan, Arvind Tomar, Lokendra Singh and Ram Kumar Dhaked

Affiliation:

Keywords: Botulinum neurotoxin, HCC subunit, renaturation, antibody isotyping, GT1b, SPR

Abstract: 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.

Cite this article as:

Kumar Singh Manglesh, Gupta Garima, Boopathi Mannan, Gupta Pallavi, Chauhan Vinita, Tomar Arvind, Singh Lokendra and Kumar Dhaked Ram, 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, Protein & Peptide Letters 2012; 19 (5) . https://dx.doi.org/10.2174/092986612800191008

DOI https://dx.doi.org/10.2174/092986612800191008	Print ISSN 0929-8665
Publisher Name Bentham Science Publisher	Online ISSN 1875-5305