FIG 1.
Construction and detection of STa-toxoid-dmLT toxoid fusions. (A) Illustration of an STa-toxoid-dmLT toxoid fusion gene. Three copies of each STa-toxoid gene were genetically fused to the 5′ end, between A1 and A2 of LTA, and the 3′ end of a monomeric dmLT peptide gene (LTR192G/L211A; as a single open reading frame) using splicing overlap extension PCRs. Primers: 1, T7-F; 2, STa-toxoid-F; 3, STa-toxoid-R; 4, LT73-R; 5, LT73-F; 6, LT211-R; 7, LT211-F; 8, T7-R. Primers 2 and 3 were used to mutate the STa gene for different STa toxoids (Table 2). Primers 1 and 3 and primers 2 and 4 were, respectively, used in two PCRs (which generated two nucleotide fragments to be fused) to produce the first segment of the fusion gene, which included the first copy of a STa toxoid and the first 75 amino acids of the LT A subunit (at the N terminus). Primers 5 and 3 and primers 2 and 6 were used in two other PCRs (for two other fragments to be fused) to generate the second segment of the fusion gene, which consisted of the second copy of a STa toxoid and amino acids 68 to 216 of the LT A subunit. Primers 7 and 3 and primers 2 and 8 were used in another two PCRs (for another two fragments to be fused) to create the third segment of the fusion gene, which contains the third copy of a STa toxoid, amino acids 204 to 240 of the LT A subunit and the LT B subunit (1 to 100 amino acids). These three segments were connected in an SOE PCR for a single open reading frame coding a STa-toxoid-dmLT toxoid fusion protein. (B) Western blot to detect each STa-toxoid-dmLT fusion protein with anti-CT antibodies. A fusion protein (100 ng) separated in 10 to 12% PAGE gel was detected using rabbit anti-CT antiserum (1:3,300; Sigma) and IRDye-labeled goat anti-rabbit IgG (1:5,000; LI-COR). (C) Western blot to detect toxoid fusion proteins with anti-STa antibodies. Fusion proteins (100 ng each) were detected with protein A-purified rabbit anti-STa antiserum (1:5,000) and IRDye-labeled goat anti-rabbit IgG (1:5,000; LI-COR). The total protein extracted from host strain 8955 was used as the negative control (−). Lane M is the protein marker (in kilodaltons; Precision Plus Protein Pre-stained standards; Bio-Rad).