title: Easy expression of the C-terminal heavy chain domain of botulinum neurotoxin serotype A as a vaccine candidate using a bi-cistronic baculovirus system.
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 (2μg) 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 10(2) LD50 botulinum A toxin with a dose as low as 0.2μg. 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.
authors: Department of Chemistry, Chung Yuan Christian University, Chungli 32023, Taiwan.
authors: Oliver B
authors: OB
authors: Villaflores
authors: Chein-Ming
authors: CM
authors: Hsei
authors: Chao-Yi
authors: CY
authors: Teng
authors: Ying-Ju
authors: YJ
authors: Chen
authors: Jiunn-Jye
authors: JJ
authors: Wey
authors: Pei-Yi
authors: PY
authors: Tsui
authors: Rong-Hwa
authors: RH
authors: Shyu
authors: Kuo-Lun
authors: KL
authors: Tung
authors: Jui-Ming
authors: JM
authors: Yeh
authors: Der-Jiang
authors: DJ
authors: Chiao
authors: Tzong-Yuan
authors: TY
authors: Wu
mesh_terms: D000818:Animals; D000907:Antibodies, Bacterial; D001428:Bacterial Vaccines / Q000276:immunology*; D016367:Baculoviridae / Q000235:genetics; D019274:Botulinum Toxins, Type A / Q000235:genetics* / Q000276:immunology*; D001906:Botulism / Q000276:immunology* / Q000517:prevention & control; D002460:Cell Line; D049452:Green Fluorescent Proteins / Q000235:genetics; D051379:Mice; D008807:Mice, Inbred BALB C; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology; D061987:Sf9 Cells; D018411:Spodoptera
chemical_list: D000907:Antibodies, Bacterial; D001428:Bacterial Vaccines; D011994:Recombinant Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D019274:Botulinum Toxins, Type A