Molecular studies on clostridium botulinum type B neurotoxin

Abstract

In this thesis, studies of Clostridium botulinum type B toxin were approached by the three sequential but methodologically different steps. For the purifcation of Clostridium botulinum type B toxin of strain Lamanna four column chromatography works including DEAE-Sephadex, Sephadex G-200 and Sephacryl S-300 were performed. The finally obtained toxin was the mixtures of nickes and unnicked form of toxin. The molecular weight of whole toxin, heavy chain and light chain of the toxin were 142 kDa, 98 kDa and 50 kDa, respectively on the SDS-PAGE. Four monoclonal antibodies were prepared and characterized using the purified toxin as an immunogen. The isotypes of the monoclonal antibidies were Ig M, Ig Gl, A Ig, Ig Gl, respectively. One of them showed high neutralizing activity and all of them were bound to the heavy chain of toxin. 1:1000 dilution of ascites fluid which was made from the one of the hybridoma cell lines was not bound to the type A toxin and could detect 0.1 ng of type B toxin on the dot immuno-binding assay. Using a commercial $\lambda$gt 11 packaging kit, a genomic library of the whole cellular DNA of C. botulinum was made. Two $\lambda$gt 11 clones of the toxin gene were identified from the library using the monoclonal antibody specific to the heavy chain of type B toxin. Neither of the expressed fusion protein from the lysogenic E. coli Y1089 showed any botulinal toxic activity. One of the clones hybridized to the oligonucleotide probe which was synthesezed according to the amino acid sequence of N-terminus of heavy chain of C. Botulinum type B. A DNA fragment (Hind III-Xba I) hybridized with the oligonucleotide probe was isolated and subcloned for the sequencing. The sequence analysis revealed that highly homologous regions exist in N-terminus of heavy chain among botulinum neurotoxins(type A, B) and tetanus toxin on the amino acid sequence level. This means that the three toxin genes are might derived from a common ancestral gene whose function i...