Glycerol phosphate buffered salt solution

Recombinant E. coli was cultured in TBY supplemented with Kn, and grown at 37°C, 225 rpm, to an OD₆₀₀ of 0.8. Protein expression was induced with 1 mM IPTG during 3h. The cells were harvested by centrifugation at 4000xg for 10 min at 4°C and resuspended in 20% glycerol/phosphate buffered salt solution (Gibco, Grand Island, NY) in 1% of the initial volume. Cell suspensions in aliquots of 2 ml were frozen quickly in a dry ice bath and stored at -80°C. Aliquots of recombinant E. coli were thawed at 4°C and 400 μl (10⁹ cells) were placed in a ball-tipped syringe for oral gavage inoculation. Groups of female C3H/HeN mice were immunized by intragastric inoculation of induced recombinant E. coli or with the empty vector (Ec) as controls. Mice received the first immunization, twice daily, for 10 days (days 1–5 and 8–12), rested for two weeks, received the first boost for 5 days (days 29–33), rested for another week and then received a second boost for 5 days (days 43–47). On day 73, mice were terminated for collection of bronchoalveolar lavage (BAL) or challenged. Low amounts of mouse blood (up to 50 μl) were obtained via tail nick on the following days: 0, 28, 42, 73, 105, 135 and 170 after immunization. Stool (GUT, gastrointestinal tract secretions), bronchoalveolar lavage (BAL) and vaginal lavage (VAL) were collected on day 73 after immunization.