Plasmid-encoded neurotoxin genes in
The present invention provides a novel isolated plasmid, wherein the plasmid is a native plasmid found in unique C. botulinum type A strains and encode either BoNT/A3 or BoNT/A4 and BoNT/B. The present invention also provides a method of obtaining a plasmid-encoded botulinum neurotoxin and botulinum neurotoxin complex comprising the step of isolating a plasmid encoding the cntA/A or cntA/B neurotoxin gene and genes encoding protein components of the toxin complex from a C. botulinum type A strain. The inventors performed comparative analyses of representative BoNT/A subtype strains by pulsed-field gel electrophoresis (PFGE) and Southern hybridizations with probes specific for the BoNT/A and B genes, cntA/A and cntA/B. Unexpectedly, the inventors determined that the genes encoding BoNT/A3 in the A3 strain, and BoNT/A4 and BoNT/B in the A4 strain, are on plasmids.
1. A method of obtaining a plasmid-encoded botulinum neurotoxin comprising the steps of:
obtaining a bacterial strain comprising a plasmid-encoded botulinum neurotoxin type A,
identifying a plasmid encoding the cntA/A or cntA/B gene from the bacterial strain, wherein (a) the cntA/A or cntA/B gene encodes BoNT/A3, and the bacterial strain is Clostridium botulinum type A strain Loch Maree; or (b) the cntA/A or cntA/B gene encodes BoNT/A4 and BoNT/B5, and the bacterial strain is Clostridium botulinum type A strain 657Ba;
separating and purifying the plasmid encoding the cntA/A or cntA/B gene from the Clostridium botulinum type A strain; and
introducing the plasmid into a clostridium bacteria and culturing the Clostridium bacteria, whereby a plasmid-encoded botulinum neurotoxin is obtained.
2. The method of claim 1 wherein the Clostridium botulinum type A strain is Loch Maree.
3. The method of claim 1 wherein the Clostridium botulinum type A strain is 657Ba.
4. A method of producing botulinum neurotoxins and botulinum neurotoxin complexes comprising the steps of:
a) isolating a plasmid encoding the cntA/A or cntA/B gene from a Clostridium botulinum type A strain, wherein (a) the cntA/A or cntA/B gene encodes BoNT/A3, and the Clostridium botulinum type A strain is Loch Maree; or (b) the cntA/A or cntA/B gene encodes BoNT/A4 and BoNT/B5, and the Clostridium botulinum type A strain is 657Ba;
b) separating and purifying the plasmid encoding the cntA/A or cntA/B gene from the Clostridium botulinum type A strain;
c) introducing the plasmid into clostridium bacteria; and
d) culturing the clostridium bacteria wherein the botulinum toxin complex and neurotoxins are produced and purified.
5. The method of claim 4 wherein the Clostridium botulinum type A strain is Loch Maree.
6. The method of claim 4 wherein the Clostridium botulinum type A strain is 657Ba.