Team:Slovenia/Results/Engineered flagellin vaccine/Engineered bacterial vaccine

B) MODIFIED BACTERIA AS MUCOSAL VACCINES

Various immunization ways have been tested for delivery of H. pylori antigens. Recent studies have shown that oral vaccination is the optimal mucosal route for induction of antigen-specific IgA response. However, antibody production has no role in protective immunity in mice. To increase the immunogenicity of H. pylori antigens, we made fusion proteins with flagellin. Studies have shown that genetically fusing an antigen to flagellin significantly increases the immunogenicity and protective potency of the antigen.

For our delivery system we selected Escherichia coli, strain JW1908-1 with mutations in genes coding for flagellin, that prevent expression of E. coli own flagellin. We cloned several constructs, which were transformed into E. coli, strain JW1908-1 (+T7 polymerase and bacterial ghost plasmid (BG)).

PROTEIN EXPRESSION

To test wheather proteins are expressed in bacteria we analyzed bacterial lysates with Western blotting and surface expression of proteins was checked with confocal microscopy.

The majority of chimeric flagellin constructs in bacteria was expressed as soluble proteins.

Western blot analysis of expressed proteins was done as indicated in Methods. Sample 1: T7-CF-UB33-CF expressed in soluble fraction of cell lysate (SN1) or in insoluble fraction (IB1); sample 3: T7-CF-AK3 expressed in soluble fraction (SN3); sample 8: TetR-CF-UB33-CF expressed in soluble fraction (SN8) and insoluble fraction (IB8); sample 9: T7-CF-pET expressed in soluble fraction (SN9) and in insoluble fraction (IB9). Expected protein sizes: T7-CF-UB33-CF and TetR-CF-UB33-CF 57.9 kDa, T7-CF-AK3 and T7-CF-pET 54.8 kDa.

With Western blot analysis we detected high expression of our proteins of expected sizes.

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