Author/Authors :
Mousavi Gargari ، Seyed Latif نويسنده Department of Biology, Faculty of Basic Sciences, Shahed University, Tehran, Iran , , Rasooli، Iraj نويسنده , , Valipour، Ebrahim نويسنده Department of Biology, Zanjan University, P.O. Box 45195-313, Zanjan, I.R. Iran , , Basiri، Mohsen نويسنده Neuroscience Research Center, Kerman University of Medical Sciences, Kerman , , Nazarian، Shahram نويسنده , , Amani، Jafar نويسنده , , Farhadi، Nima نويسنده Department of Biology, Institute of Basic Sciences, Imam-Hossein University, P.O. Box 16575-347, Tehran, I.R. Iran ,
Abstract :
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 C-terminal 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 mg 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.
