CLONING AND SEQUENCING OF A HEMAGGLUTININ COMPONENT OF THE BOTULINUM NEUROTOXIN COMPLEX ENCODED BY CLOSTRIDIUM-BOTULINUM TYPE-A AND TYPE-B

Degenerate oligonucleotide primers designed to the N-terminal amino acid sequence of proteins purified from the botulinum neurotoxin complex were used to amplify DNA fragments using PCR from genomic DNA of Clostridium botulinum types A: NCTC 7272, and B: NCTC 7273 (proteolytic strain) and Eklund 17B (non-proteolytic strain). A fragment of similar to 1.9 kb was amplified using template DNA from each strain, which was subsequently cloned in E. coli and the sequences determined. Two open reading frames (orfs) were discovered: one corresponding to a protein similar to 33.7 kD which shows between 36 and 41% identity with the major hemagglutinin component (HA-33) of the botulinum progenitor complex encoded by C. botulinum type C. The second orf, encoding a protein of similar to 21.7 kD, P-21, of 178 amino acids (179 in strain NCTC 7273) has similar to 93% identity between the type A and two type B strains, but is absent from type C.