• Journal of Microbiology and Biotechnology
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• v.13 no.2
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• pp.287-291
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• 2003

Botulinum neurotoxin type A (BONT/A) is an extremely potent toxin, which is produced by Clostridium botulinum. The light chain of this protein (BONT/A LC), which is known as a zinc endopeptidase, cleaves SNAP-25 involved in the exocytosis process. In this work, the expression of recombinant BoNT/A LC in E. coli is described. The BONT/A LC gene of C. botulinum contains a high frequency of the arginine AGA and isoleucine ATA codons that are rarely used in genes of E. coli, hampering the translation of recombinant protein. The argD and ilex tRNA genes were cloned into pACYC184 vector, resulting in pAAD131X plasmid. The translational stress of the toxin gene related to codon bias was reversed by fupplernentation of the AGA arginyl tRNA of T4 phage and AUA isoleucyl tRNA of E. coli. This system may be applicable for the expression of a variety of AT-rich heterologous genes in E. coli.

• The Korean Journal of Pain
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• v.35 no.4
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• pp.391-402
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• 2022

Background: The mechanism of peripheral axon transport in neuropathic pain is still unclear. Chemokine ligand 13 (CXCL13) and its receptor (C-X-C chemokine receptor type 5, CXCR5) as well as GABA transporter 1 (GAT-1) play an important role in the development of pain. The aim of this study was to explore the axonal transport of CXCL13/CXCR5 and GAT-1 with the aid of the analgesic effect of botulinum toxin type A (BTX-A) in rats. Methods: Chronic constriction injury (CCI) rat models were established. BTX-A was administered to rats through subcutaneous injection in the hind paw. The pain behaviors in CCI rats were measured by paw withdrawal threshold and paw withdrawal latencies. The levels of CXCL13/CXCR5 and GAT-1 were measured by western blots. Results: The subcutaneous injection of BTX-A relieved the mechanical allodynia and heat hyperalgesia induced by CCI surgery and reversed the overexpression of CXCL13/CXCR5 and GAT-1 in the spinal cord, dorsal root ganglia (DRG), sciatic nerve, and plantar skin in CCI rats. After 10 mmol/L colchicine blocked the axon transport of sciatic nerve, the inhibitory effect of BTX-A disappeared, and the levels of CXCL13/CXCR5 and GAT-1 in the spinal cord and DRG were reduced in CCI rats. Conclusions: BTX-A regulated the levels of CXCL13/CXCR5 and GAT-1 in the spine and DRG through axonal transport. Chemokines (such as CXCL13) may be transported from the injury site to the spine or DRG through axonal transport. Axon molecular transport may be a target to enhance pain management in neuropathic pain.

• Journal of Microbiology and Biotechnology
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• v.21 no.7
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• pp.719-727
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• 2011

Botulism is a neuroparalytic disease caused by Clostridium botulinum, which produces seven (A-G) antigenically diverse neurotoxins (BoNTs). BoNTs are the most poisonous substances known to humans, with a median lethal dose ($LD_{50}$) of approximately 1 ng/kg of body weight. Owing to their extreme potency and lethality, they have the potential to be used as a bioterrorism agent. The mouse bioassay is the gold standard for the detection of botulinum neurotoxins; however, it requires at least 3-4 days for completion. Attempts have been made to develop an ELISA-based detection system, which is potentially an easier and more rapid method of botulinum neurotoxin detection. The present study was designed using a synthetic gene approach. The synthetic gene encoding the catalytic domain of BoNT serotype B from amino acids 1-450 was constructed with PCR overlapping primers (BoNT/B LC), cloned in a pQE30 UA vector, and expressed in an E. coli M15 host system. Recombinant protein production was optimized at 0.5 mM IPTG final concentration, 4 h post induction, resulting in a maximum yield of recombinant proteins. The immunogenic nature of the recombinant BoNT/B LC protein was evaluated by ELISA. Antibodies were raised in BALB/c mice using various adjuvants. A significant rise in antibody titer (p<0.05) was observed in the Alum group, followed by the Titermax Classic group, Freund's adjuvant, and the Titermax Gold group. These developed high-titer antibodies may prove useful for the detection of botulinum neurotoxins in food and clinical samples.

• The Korean Journal of Physiology and Pharmacology
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• v.19 no.4
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• pp.349-355
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• 2015

We examined the effects of peripherally or centrally administered botulinum neurotoxin type A (BoNT-A) on orofacial inflammatory pain to evaluate the antinociceptive effect of BoNT-A and its underlying mechanisms. The experiments were carried out on male Sprague-Dawley rats. Subcutaneous (3 U/kg) or intracisternal (0.3 or 1 U/kg) administration of BoNT-A significantly inhibited the formalin-induced nociceptive response in the second phase. Both subcutaneous (1 or 3 U/kg) and intracisternal (0.3 or 1 U/kg) injection of BoNT-A increased the latency of head withdrawal response in the complete Freund's adjuvant (CFA)-treated rats. Intracisternal administration of N-methyl-D-aspartate (NMDA) evoked nociceptive behavior via the activation of trigeminal neurons, which was attenuated by the subcutaneous or intracisternal injection of BoNT-A. Intracisternal injection of NMDA up-regulated c-Fos expression in the trigeminal neurons of the medullary dorsal horn. Subcutaneous (3 U/kg) or intracisternal (1 U/kg) administration of BoNT-A significantly reduced the number of c-Fos immunoreactive neurons in the NMDA-treated rats. These results suggest that the central antinociceptive effects the peripherally or centrally administered BoNT-A are mediated by transcytosed BoNT-A or direct inhibition of trigeminal neurons. Our data suggest that central targets of BoNT-A might provide a new therapeutic tool for the treatment of orofacial chronic pain conditions.

• Journal of the Korean Society of Laryngology, Phoniatrics and Logopedics
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• v.18 no.1
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• pp.51-55
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• 2007

Objectives: The aim of this study is to compare the efficacy of fresh versus frozen reconstituted botulinum toxin type A (BTX-A) for the treatment of adductor type spasmodic dysphonia. Materials and Methods: After reconstitution with normal saline, BTX-A was used within 4 hours or it was kept frozen in a consumer grade freezer at about $-25^{\circ}C$ for up to 4 months. Thirty patients with spasmodic dysphonia were randomly assigned and treated with the either fresh or frozen BTX-A. About 83% of injections resulted in a satisfactory outcome with 5.3 months of mean action duration. Treatment outcomes and side effects of total 161 injections were compared along the duration of keeping BTX-A frozen. Results: There were no statistical differences in the duration of action, self-rated satisfaction score, and the duration of hoarseness and/or aspiration between fresh and frozen BTX-A treated groups. No significant side effects were observed and the frozen BTX-A were proved to be free of bacterial contamination. Conclusion: After being reconstituted and kept frozen, BTX-A may be safely used for more than 4 months without significant loss of its effectiveness or additional side effects.

• Journal of Microbiology and Biotechnology
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• v.29 no.7
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• pp.1165-1176
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• 2019

Botulinum neurotoxins (BoNTs), produced by Clostridium botulinum, are the most toxic substances known. However, the number of currently approved medical countermeasures for these toxins is very limited. Therefore, studies on therapeutic antitoxins are essential to prepare for toxin-related emergencies. Currently, more than 10,000 Halla horses, a crossbreed between the native Jeju and Thoroughbred horses, are being raised in Jeju Island of Korea. They can be used for equine antitoxin experiments and production of hyperimmune serum against BoNT/A1. Instead of the inactivated BoNT/A1 toxoid, Halla horse was immunized with the receptor-binding domain present in the C-terminus of heavy chain of BoNT/A1 (BoNT/A1-HCR) expressed in Escherichia coli. The anti-BoNT/A1-HCR antibody titer increased rapidly by week 4, and this level was maintained for several weeks after boosting immunization. Notably, $20{\mu}l$ of the week-24 BoNT/A1-HCR(-immunized) equine serum showed an in vitro neutralizing activity of over 8 international units (IU) of a reference equine antitoxin. Furthermore, $20{\mu}l$ of equine serum and $100{\mu}g$ of purified equine $F(ab^{\prime})_2$ showed 100% neutralization of 10,000 $LD_{50}$ in vivo. The results of this study shall contribute towards optimizing antitoxin production for BoNT/A1, which is essential for emergency preparedness and response.