Expression and Characterization of Synthetic Heat-Labile Enterotoxin B Subunit and Hemagglutinin-Neuraminidase-Neutralizing Epitope Fusion Protein in Escherichia coli and Tobacco Chloroplasts

Synthetic enterotoxigenic Escherichia coli heat-labile enterotoxin B subunit-HN-neutralizing epitope fusion protein was expressed in E. coli and tobacco chloroplasts. Bacterial and chloroplastic recombinant LTB-HN-neutralizing epitope (LTB-HNE) fusion proteins showed the pentameric structures by sodium dodecyl sulfate polyacrylamide gel electrophoresis and a strong affinity for G(M1)-ganglioside. Bacterial and chloroplastic recombinant LTB-HNE was detected by Western blot analysis using polyclonal antibodies to HN-neutralizing epitope. Insertion of the gene encoding LTB-HNE protein into the chloroplast genomic DNA of spectinomycin-resistant plants was confirmed by polymerase chain reaction. The presence of the LTB-HNE specific transcript in the total RNA of transgenic plant leaves was verified by reverse transcriptase polymerase chain reaction. The highest level of expression of recombinant LTB-HNE fusion proteins in the leaves of transplastomic plants was about 0.5% of the total soluble protein. Growth rates, flowering, and seed setting were not affected, and the cassette for homologous integration and gene expression in the transplastomic T1 plant was subsequently inherited.