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.
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.
authors.affiliation: Biotechnology Division, Protective Device Division, Defence Research & Development Establishment, Gwalior-474002, India.
authors.forename: Manglesh Kumar
authors.initials: MK
authors.lastname: Singh
authors.forename: Garima
authors.initials: G
authors.lastname: Gupta
authors.forename: Mannan
authors.initials: M
authors.lastname: Boopathi
authors.forename: Pallavi
authors.initials: P
authors.lastname: Gupta
authors.forename: Vinita
authors.initials: V
authors.lastname: Chauhan
authors.forename: Arvind
authors.initials: A
authors.lastname: Tomar
authors.forename: Lokendra
authors.initials: L
authors.lastname: Singh
authors.forename: Ram Kumar
authors.initials: RK
authors.lastname: Dhaked
mesh_terms: D000818:Animals; D000907:Antibodies, Bacterial / Q000097:blood / Q000276:immunology; D001428:Bacterial Vaccines / Q000235:genetics / Q000276:immunology; D001665:Binding Sites; D019274:Botulinum Toxins, Type A / Q000737:chemistry / Q000235:genetics* / Q000276:immunology* / Q000378:metabolism; D003001:Cloning, Molecular; D003014:Clostridium botulinum / Q000235:genetics* / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005260:Female; D005732:Gangliosides / Q000378:metabolism; D006639:Histidine; D007074:Immunoglobulin G / Q000097:blood / Q000276:immunology; D007075:Immunoglobulin M / Q000097:blood / Q000276:immunology; D051379:Mice; D009842:Oligopeptides; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D017384:Sequence Deletion; D020349:Surface Plasmon Resonance
chemical_list: D000907:Antibodies, Bacterial; D001428:Bacterial Vaccines; D005732:Gangliosides; C471213:His-His-His-His-His-His; D007074:Immunoglobulin G; D007075:Immunoglobulin M; D009842:Oligopeptides; D011994:Recombinant Proteins; D006639:Histidine; C016481:trisialoganglioside GT1; D019274:Botulinum Toxins, Type A