Expression of a Cholera Toxin B Subunit-Neutralizing Epitope of the Porcine Epidemic Diarrhea Virus Fusion Gene in Transgenic Lettuce (Lactuca sativa L.)

摘要

Transgenic plants have been used as a safe and economic expression system for the production of edible vaccines. A synthetic cholera toxin B subunit gene (CTB) was fused with a synthetic neutralizing epitope gene of the porcine epidemic diarrhea virus (sCTB–sCOE), and the sCTB–sCOE fusion gene was introduced into a plant expression vector under the control of the ubiquitin promoter. This plant expression vector was transformed into lettuce (Lactuca sativa L.) using the Agrobacterium-mediated transformation method. Stable integration and transcriptional expression of the sCTB–sCOE fusion gene was confirmed using genomic DNA PCR analysis and northern blot analysis, respectively. The results of western blot analysis with anti-cholera toxin and anti-COE antibody showed the synthesis and assembly of CTB–COE fusion protein into oligomeric structures with pentameric sizing. The biological activity of CTB–COE fusion protein to its receptor, GM1-ganglioside, in transgenic plants was confirmed via GM1-ELISA with anti-cholera toxin and anti-COE antibody. Based on GM1-ELISA, the expression level of CTB–COE fusion proteins reached 0.0065% of the total soluble protein in transgenic lettuce leaf tissues. Transgenic lettuce successfully expressing CTB–COE fusion protein will be tested to induce efficient immune responses against porcine epidemic diarrhea virus infection by administration with raw material.