Use of Recombinant DNA Techniques for the Production of a More Effective Anthrax Vaccine
Abstract
We report an improved isolation procedure for the preparation of pX01 and pX02 plasmids of B. anthracis. These plasmids have been physically characterized with regard to buoyant density, GC content and size analysis using restriction enzyme digestions. Restriction maps of these DNAs have been generated. pX01 is 175 kbp and pX02 is 95 kbp. The location of the toxin genes, protective antigen (PA), lethal factor (LF) and edema factor (EF), have been positioned on pX01. The EF gene has been cloned and sequenced. Unique features of the EF gene include a putative ATP binding site. Expression vectors in E. coli have been used to produce large quantities of this protein. Expression vectors for LF and PA in both E. and coli and B. subtilis have been used for enhanced expression of these genes as well. In order to generate a safe anthrax vaccine using recombinant DNA techniques, we have begun experiments to specifically mutant each of these toxin genes to generate non-functional proteins that retain most, if not all, of their immunogenic properties. For example, the trypsin cleavage site of PA is being altered to remove the Arg-Lys- Lys-Arg sequence which is cleaved to activate PA. In addition, the putative ATP binding site of EF is being mutated to prevent enzyme activity. These experiments should generate mutant toxin proteins which will be safe vaccine components which will then be tested in animals.
Document Details
- Document Type
- Technical Report
- Publication Date
- Jun 30, 1987
- Accession Number
- ADA187206
Entities
People
- Donald L. Robertson
Organizations
- Brigham Young University