Document Type: Research Paper
Department of Biology, Shahed University, P.O. Box 18151-159, Tehran, I.R. Iran.
Department of Biology, Zanjan University, P.O. Box 45195-313, Zanjan, I.R. Iran.
Department of Biology, Institute of Basic Sciences, Imam-Hossein University, P.O. Box 16575-347, Tehran, I.R. Iran.
Medical Biology Research Center, Baqiyatallah University of Medical Sciences, P.O. Box 19395-5487, Tehran, I.R. Iran.
Clostridium Botulinum Type E neurotoxin heavy chain consists of two domains: the translocation domain as
the N-terminal half and the binding domain as the Cterminal half (Hc). One effective way to neutralize botulinum neurotoxin is to inhibit binding of this toxin to neuromuscular synapses with antibodies against binding domain. Two synthetic genes, coding for Hc (the full length binding domain) and the c-terminal quarter of binding domain (HcQ), were cloned in pET-28a vector and over-expressed in E. coli BL21 (DE3) cells.
These recombinant proteins were purified by affinity Ni-NTA column (under native condition). Mice were
vaccinated with 2 μg of purified proteins, respectively; at step one with complete adjuvant, steps two and
three with incomplete adjuvant and step four only with phosphate buffered saline (PBS). Enzyme-linked
immunosorbent assay (ELISA) has been performed with mice serum samples 14 days following their third
and final vaccination. Binding activity of the purified proteins to ganglioside and synaptotagmin II was analyzed by ELISA. The results showed that HcQ and Hc could bind with ganglioside. Based on challenge
experiments it was revealed that HcQ, Hc and BoNT/E toxoid could give protections in mice challenged with
102 , 104 and 105 minimum lethal dose (MLD) dose of BoNT/E.