PMID- 23313783
OWN - NLM
STAT- MEDLINE
DA  - 20130325
DCOM- 20131017
IS  - 1879-0984 (Electronic)
IS  - 0166-0934 (Linking)
VI  - 189
IP  - 1
DP  - 2013 Apr
TI  - Easy expression of the C-terminal heavy chain domain of botulinum neurotoxin
      serotype A as a vaccine candidate using a bi-cistronic baculovirus system.
PG  - 58-64
LID - 10.1016/j.jviromet.2012.11.035 [doi]
LID - S0166-0934(12)00427-2 [pii]
AB  - 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 (2mug) 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 0.2mug. 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.
CI  - Copyright (c) 2012 Elsevier B.V. All rights reserved.
AD  - Department of Chemistry, Chung Yuan Christian University, Chungli 32023, Taiwan.
FAU - Villaflores, Oliver B
AU  - Villaflores OB
FAU - Hsei, Chein-Ming
AU  - Hsei CM
FAU - Teng, Chao-Yi
AU  - Teng CY
FAU - Chen, Ying-Ju
AU  - Chen YJ
FAU - Wey, Jiunn-Jye
AU  - Wey JJ
FAU - Tsui, Pei-Yi
AU  - Tsui PY
FAU - Shyu, Rong-Hwa
AU  - Shyu RH
FAU - Tung, Kuo-Lun
AU  - Tung KL
FAU - Yeh, Jui-Ming
AU  - Yeh JM
FAU - Chiao, Der-Jiang
AU  - Chiao DJ
FAU - Wu, Tzong-Yuan
AU  - Wu TY
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
DEP - 20130111
PL  - Netherlands
TA  - J Virol Methods
JT  - Journal of virological methods
JID - 8005839
RN  - 0 (Antibodies, Bacterial)
RN  - 0 (Bacterial Vaccines)
RN  - 0 (Recombinant Proteins)
RN  - 0 (enhanced green fluorescent protein)
RN  - 147336-22-9 (Green Fluorescent Proteins)
RN  - EC 3.4.24.69 (Botulinum Toxins, Type A)
SB  - IM
MH  - Animals
MH  - Antibodies, Bacterial
MH  - Bacterial Vaccines/*immunology
MH  - Baculoviridae/genetics
MH  - Botulinum Toxins, Type A/*genetics/*immunology
MH  - Botulism/*immunology/prevention & control
MH  - Cell Line
MH  - Green Fluorescent Proteins/genetics
MH  - Mice
MH  - Mice, Inbred BALB C
MH  - Recombinant Proteins/genetics/immunology
MH  - Sf9 Cells
MH  - Spodoptera
EDAT- 2013/01/15 06:00
MHDA- 2013/10/18 06:00
CRDT- 2013/01/15 06:00
PHST- 2011/10/08 [received]
PHST- 2012/11/18 [revised]
PHST- 2012/11/21 [accepted]
PHST- 2013/01/11 [aheadofprint]
AID - S0166-0934(12)00427-2 [pii]
AID - 10.1016/j.jviromet.2012.11.035 [doi]
PST - ppublish
SO  - J Virol Methods. 2013 Apr;189(1):58-64. doi: 10.1016/j.jviromet.2012.11.035. Epub
      2013 Jan 11.