Nucleotide sequencing and expression of clostridium botulinum neurotoxin type B in escherichia coli

Abstract

The nucleotide sequences of the cloned Clostridium botulinum type B neurotoxin (BoNT/B) gene fragments were analyzed by dideoxy chain termination methods. The 2.5 Kb fragment coded for the C- terminus of L chain and H chain fragment. The nucleotide sequence for the BoNT/B fragment was determined, and derived amono acids sequence compared with those of botulinum neurotoxin type A (BoNT/A) and tetanus toxin (TeTx). It was found that there exist highly homologous regions and specially extremely conserved trp amino acid. 2.4 Kb HindIII fragment (containing N-terminus of H chain) and 1.9 Kb Xbal fragmant (containing not) were expressed in E. coli with pRSET, a plasmid vector containing the T7 promoter and a portion of the mbs gene. The fusion protein containing N-terminus of H chain were shown to react with, anti-BoNT/B monoclonal antibody BTBM4 recognizing H chain. This result suggests that the epitope of BTBM4 is located in N-terminus of H chain. With previous works (Davis and Hassen, 1988, Ham and Homsy, 1988). But the diffusivity values were larger by a factor of 5 since the self-sharpening effect is considered in this study.