Production of FaeG, the major subunit of K88 fimbriae, in transgenic tobacco plants and its immunogenicity in mice

Abstract

Transgenic tobacco plants stably expressing recombinant FaeG, which is the major subunit and adhesin of K88ad fimbriae, were obtained. Analysis of sera from immunized mice indicates that in mice, the immunogenicity induced by plant-derived FaeG protein is comparable to that generated with traditional approaches.

FIG. 1.

Analysis of the expression of faeG in transgenic plants. (A) Detection (using RT-PCR) of faeG transcription in transgenic plants. RT-PCR was performed (using specific primers that amplify a 789-bp DNA fragment of faeG) with total RNA from leaves of transgenic tobaccos (32). Lane 1, λDNA (digested with HindIII/EcoRI) molecular weight marker; lanes 2 and 3, RNA from transgenic tobacco; lanes 4 and 5, PCR amplification without RT reaction as a control for DNA contamination; lane 6, RNA from nontransgenic tobacco. (B) Immunoblot detection of recombinant FaeG synthesized in transgenic tobacco. TSP from tobacco leaves was fractionated by sodium dodecyl sulfate-15% polyacrylamide gel electrophoresis. The recombinant FaeG protein was detected with anti-FaeG antibody as the primary antibody and alkaline phosphatase-conjugated goat anti-rabbit IgG as the secondary antibody. Lane 1, 0.5 μg of purified recombinant FaeG expressed in E. coli BL₂₁(DE₃+K88) as a positive control; lanes 2 and 3, 100 μg each of TSP from transgenic tobacco plants; lane 4, 100 μg of TSP from nontransgenic tobacco.

FIG. 2.

Immunoblot analysis of the expression level of recombinant FaeG in T₀, T₁, and T₂ generations of transgenic tobacco plants. A total of 100 μg of TSP was loaded for each lane. Lane 1, a nontransgenic plant; lanes 2 and 3, T₀ transgenic plants; lanes 4 and 5, T₁ transgenic plants; lanes 6 and 7, T₂ transgenic plants.

FIG. 3.

Immunogenicity of recombinant FaeG from transgenic tobacco. (A) Mice were immunized intraperitoneally on days 0, 14, 28, and 42 with either purified K88ad fimbriae or crude extracts from transgenic tobacco leaves. Arrows indicate the inoculating schedule. (B) Anti-FaeG antibodies detected by immunoblot analysis. ETEC K88ad fimbriae and purified recombinant FaeG from E. coli BL₂₁(DE₃+K88) were loaded in a well for sodium dodecyl sulfate-15% polyacrylamide gel electrophoresis and transferred to nitrocellulose membrane. After the reaction mixtures were incubated with the sera of mice immunized with purified fimbriae, transgenic tobacco leaf extracts, or nontransgenic tobacco leaf extracts and then washed three more times, the reactions were developed by the addition of the substrate nitroblue tetrazolium-5-bromo-4-chloro-3-indolylphosphate. Lanes 1 and 2, serum from a mouse immunized with purified fimbriae as a positive control; lanes 3 and 4, serum from a mouse immunized with transgenic tobacco extracts; lanes 5 and 6, serum from a mouse immunized with nontransgenic tobacco extracts as a negative control. Lanes 1, 3, and 5, 0.5 μg of purified K88ad fimbriae; lanes 2, 4, and 6, 0.5 μg of purified recombinant FaeG from E. coli BL₂₁(DE₃+K88).