Easy expression of the C-terminal heavy chain domain of botulinum neurotoxin serotype A as a vaccine candidate using a bi-cistronic baculovirus system
Journal
Journal of Virological Methods
Journal Volume
189
Journal Issue
1
Pages
58-64
Date Issued
2013
Author(s)
Villaflores O.B.
Hsei C.-M.
Teng C.-Y.
Chen Y.-J.
Wey J.-J.
Tsui P.-Y.
Shyu R.-H.
Yeh J.-M.
Chiao D.-J.
Wu T.-Y.
Abstract
Clostridial botulinum neurotoxin (BoNT) is one of the most toxic proteins causing the food borne disease, botulism. In previous studies, recombinant BoNT production by Escherichia coli and yeast Pichia pastoris has been hampered by high AT content and codon bias in the gene encoding BoNT and required a synthetic gene to resolve this intrinsic bottleneck. This paper reports the simultaneous expression of the C-terminal heavy chain domain of BoNT (rBoNT/A-HC-6h) and enhanced green fluorescent protein (EGFP) using a bi-cistronic baculovirus-insect cell expression system. The expression of EGFP facilitated the monitoring of viral infection, virus titer determination, and isolation of the recombinant virus. Protein fusion with hexa-His-tag and one-step immobilized metal-ion affinity chromatography (IMAC) purification produced a homogenous, stable, and immunologically active 55-kDa rBoNT/A-HC-6h (about 3mg/L) with >90% purity. Furthermore, measured levels of serum titers were 8-folds for mice vaccinated with the purified rBoNT/A-HC-6h (2£gg) than for mice administered with botulinum toxoid after initial immunization. Challenge experiment with botulinum A toxin demonstrated the immunoprotective activity of purified rBoNT/A-HC-6h providing the mice full protection against 102 LD50 botulinum A toxin with a dose as low as 0.2£gg. This study provided supportive evidence for the use of a bi-cistronic baculovirus-Sf21 insect cell expression system in the facile expression of an immunogenically active rBoNT/A-HC. ? 2012 Elsevier B.V.
Subjects
Baculovirus
Botulinum toxin serotype A
Internal ribosome entry site
Rhopalosiphum padi virus
Vaccine
SDGs
Type
journal article