Poster: Emerging technologies
Abs #
908: Expression of Bacillus anthracis protective antigen in transgenic chloroplasts of tobacco, a non-food/feed crop
|
|
Presenter: |
Koya, Vijay , vkoya@mail.ucf.edu |
Authors | Koya, Vijay (A) Daniell, Henry (A) | | Affiliations: |
(A): University of Central Florida
|
|
|
The CDC lists Bacillus anthracis as a category A agent and estimates the cost of an anthrax attack to be $26 billion per 100,000 exposed individuals. Concerns regarding anthrax vaccine purity, a requirement for multiple injections, and a limited supply of the protective antigen, underscore the urgent need for an improved vaccine. Therefore, the 83 kDa immunogenic B. anthracis protective antigen (PA) was expressed in transgenic tobacco chloroplasts. The PA gene (pag) was cloned into a chloroplast vector along with the psbA regulatory signals to enhance translation. Chloroplast integration of the transgenes was confirmed by PCR and Southern blot analyses. Crude plant extracts contained up to 2.5 mg full length PA/g of fresh leaf tissue under continuous illumination in mature leaves and this showed exceptional stability for several months in stored leaves or crude extracts. Co-expression of the ORF 2 chaperonin from B. thuringiensis did not increase PA accumulation or induce folding into cuboidal crystals in transgenic chloroplasts. Trypsin, chymotrypsin and furin proteolytic cleavage sites present in PA were protected in transgenic chloroplasts because only full length PA 83 was observed without any degradation products. Both CHAPS and SDS detergents extracted PA with equal efficiency and PA was observed in the soluble fraction. Chloroplast-derived PA was functionally active in lysing mouse macrophages when combined with lethal factor. Crude leaf extracts contained up to 25 μg functional PA/ml. With an average yield of 172 mg of PA per Petit Havana plant (an experimental cultivar) grown in a greenhouse, 400 million doses of vaccine (free of contaminants) could be produced per acre; this could be further enhanced 18-fold using a commercial cultivar in the field.
