Mining the Francisella tularensis proteome for vaccine candidates
Date
2012
Authors
White, Kimberly G., author
Belisle, John, advisor
Schweizer, Herbert, committee member
Tjalkens, Ron, committee member
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Abstract
Based on methodologies developed for the identification of T cell antigen of other intracellular bacterial pathogens, a proteomic approach was applied for the elucidation of T cell antigens of Francisella tularensis (Covert, 2001). Specifically, subcellular components (membrane and soluble) of F. tularensis LVS were resolved by size using preparative SDS-PAGE and fractions collected using a whole gel elution technique. A total of 16 soluble and 19 membrane-sized fractions were produced, each of which were assessed for antigen reactivity based on the ability to elicit IFN-γ; from splenocytes of F. tularensis LVS-infected mice. Of these 35 preparative SDS-PAGE fractions, seven yielded a dominant T cell response. These seven fractions were further investigated using tandem mass spectrometry (MS/MS) to identify individual proteins in each immunodominant fraction. A total of 40 and 31 proteins were identified with greater than 95% confidence from the immunodominant membrane and soluble fractions, respectively. Further, MS/MS analysis of different protein quantities (2.5 μg to 10 μg) allowed for identification of the most abundant proteins in each fraction, thus focusing the number of possible proteins to nine proteins of interest. These data provide the basis for production of recombinant proteins and further immunological evaluations to select suitable candidates for inclusion in a subunit vaccine against tularemia.
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Subject
Francisella tularensis LVS
murine
proteome
subunit
tularemia
vaccine