title: Expression of a cholera toxin B subunit-neutralizing epitope of the porcine epidemic diarrhea virus fusion gene in transgenic lettuce (Lactuca sativa L.).
abstract: 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, G(M1)-ganglioside, in transgenic plants was confirmed via G(M1)-ELISA with anti-cholera toxin and anti-COE antibody. Based on G(M1)-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.
authors.affiliation: Department of Molecular Biology, Chonbuk National University, Jeonju, Chonbuk 561-756, Republic of Korea.
authors.forename: Nguyen-Xuan
authors.initials: NX
authors.lastname: Huy
authors.forename: Moon-Sik
authors.initials: MS
authors.lastname: Yang
authors.forename: Tae-Geum
authors.initials: TG
authors.lastname: Kim
mesh_terms: D015152:Blotting, Northern; D015153:Blotting, Western; D002772:Cholera Toxin / Q000096:biosynthesis* / Q000235:genetics; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000235:genetics; D005677:G(M1) Ganglioside / Q000378:metabolism; D015151:Immunoblotting; D018545:Lactuca / Q000235:genetics* / Q000378:metabolism; D030821:Plants, Genetically Modified / Q000235:genetics* / Q000378:metabolism; D016133:Polymerase Chain Reaction; D053485:Porcine epidemic diarrhea virus / Q000235:genetics* / Q000276:immunology; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D022462:Vaccines, Edible / Q000096:biosynthesis* / Q000235:genetics; D014760:Viral Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics
chemical_list: D000939:Epitopes; D011994:Recombinant Proteins; D022462:Vaccines, Edible; D014760:Viral Fusion Proteins; D005677:G(M1) Ganglioside; D002772:Cholera Toxin