title: Cloning, high level expression and immunogenicity of 1163-1256 residues of C-terminal heavy chain of C. botulinum neurotoxin type E.
abstract: Botulinum neurotoxins (BoNTs) inhibit neurotransmitter release from peripheral cholinergic synapses. BoNTs consist of a toxifying light chain and a heavy chain (HC) linked through a disulfide bond. In the present study we explored the immunogenicity and protective capability of the most effective part corresponding to 1163-1256 residues of botulinum type E neurotoxin HC gene. DNA encoding the 93 C-terminal amino acid of HC residues was synthesized with optimal codon usage for expression. These DNA fragments were ligated into a pLivSelect vector and subcloned into expression vector pET32a. Recombinant plasmids were then transformed into Escherichia coli strain BL21 DE3. The recombinant protein was purified by nickel affinity gel column chromatography. The HC1163-1256 was identified by antibodies raised against BoNT/E. HC1163-1256 was shown to bind with synaptotagmin and gangliosides, indicating that the expressed and purified HC1163-1256 protein retains a functionally active conformation. The immunization with recombinant protein induced a protection level in mice. The immunization with 2mug of the recombinant protein induced a significant protection level in mice. In conclusion, availability of the recombinant protein provides an effective system to study the biochemical and physical interactions involved during BoNT binding to nerve cells and protection against its toxicity.
authors.affiliation: Medical Biology Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran.
authors.forename: Abdoulreza Agheli
authors.initials: AA
authors.lastname: Mansour
authors.forename: Seyed Latif
authors.initials: SL
authors.lastname: Mousavi
authors.forename: Iraj
authors.initials: I
authors.lastname: Rasooli
authors.forename: Shahram
authors.initials: S
authors.lastname: Nazarian
authors.forename: Jafar
authors.initials: J
authors.lastname: Amani
authors.forename: Nima
authors.initials: N
authors.lastname: Farhadi
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000917:Antibody Formation / Q000502:physiology; D001428:Bacterial Vaccines / Q000737:chemistry / Q000235:genetics / Q000627:therapeutic use; D001483:Base Sequence; D001905:Botulinum Toxins / Q000737:chemistry / Q000235:genetics* / Q000276:immunology*; D001906:Botulism / Q000097:blood / Q000276:immunology / Q000517:prevention & control; D003001:Cloning, Molecular; D047970:Clostridium botulinum type E / Q000276:immunology; D015870:Gene Expression; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary / Q000235:genetics; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D014169:Transformation, Bacterial / Q000502:physiology; D014614:Vaccines, Synthetic / Q000737:chemistry / Q000235:genetics / Q000627:therapeutic use
chemical_list: D001428:Bacterial Vaccines; D021122:Protein Subunits; D014614:Vaccines, Synthetic; D001905:Botulinum Toxins; C096326:botulinum toxin type E