Expression and assembly of ApxIIA toxin of Actinobacillus pleuropneumoniae fused with the enterotoxigenic E. coli heat-labile toxin B subunit in transgenic tobacco

Actinobacillus pleuropneumoniae is a major etiological agent of swine pleuropneumonia, a highly contagious respiratory infection that causes severe economic losses in the swine production industry. ApxIIA, one of the virulence factors in A. pleuropneumoniae, is considered a candidate for the development of a vaccine against this bacterial infection. In this study, a fusion gene consisting of enterotoxigenic Escherichia coli heat-labile enterotoxin B subunit (LTB) and an ApxIIA fragment (amino acids 619-801) (ApxIIA(619-801)) was fused to the endoplasmic reticulum (ER) retention signal (SEKDEL) and introduced into a plant expression vector under the control of a ubiquitin promoter. The plant expression vector was transformed into tobacco Nicotiana tabacum L. cv. MD609 using an Agrobacterium-mediated transformation procedure. The integration and transcription of the LTB fusion gene were confirmed by genomic DNA PCR amplification and Northern blot analysis, respectively. The synthesis and assembly of LTB fusion protein were evidenced by Western blot analysis and the G(M1)-ganglioside enzyme-linked immunosorbent assay (G(M1)-ELISA). A quantitative ELISA used to measure the amount of LTB fusion protein produced in the transgenic plant revealed high levels of the fusion protein, up to 30 mu g g(-1), in lyophilized leaf tissue. These results demonstrate the feasibility of using a transgenic plant to express the LTB-ApxIIA(619-801) fusion protein as a first step towards producing plant-based edible vaccines to elicit immune responses against A. pleuropneumoniae via an oral mucosal delivery system.