[Modification and high level expression of protective antigen fragment of botulinum neurotoxin serotype A]

Designed a pair of primers through modifying N-terminal bases (5bps) of gene after ATG but not changing amino acid, and amplified a smaller mutated gene sequnce (468bp) containing two protective antigenic determinants from pBlue-BoNTaHc, N-terminal codon of mutated gene fragment is changed from low to high frenqence in E. coli. Mutated gene was ligated into pGEM-T vector and sequenced, then, cloned into a expression plasmid pBV220. As a result, cloned gene was expressed in insoluble form by temperature inducing (from 30 degrees C to 42 degrees C) in E. coli. Expression product is 40% of total proteins and is of specific binding activity to antibody in ELISA. The successful modification and high level expression of protective fragment of botulinum neurotoxin serotype A(BoNTaHc468) gene is conducive to further study on antitoxin and vaccine.