Cloning, high level expression and immunogenicity of 1163-1256 residues of C-terminal heavy chain of C. botulinum neurotoxin type E
- نوع فایل : کتاب
- زبان : انگلیسی
- مؤلف : Abdoulreza Agheli Mansour a, Seyed Latif Mousavi b,*, Iraj Rasooli b, Shahram Nazarian c, Jafar Amani a, Nima Farhadi a
- چاپ و سال / کشور: 2010
Description
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 2 mg 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.
Biologicals 38 (2010) 260–264 Article history: Received 31 May 2009 Received in revised form 29 September 2009 Accepted 29 September 2009
