Production of a heat-labile enterotoxin B subunit-porcine epidemic diarrhea virus-neutralizing epitope fusion protein in transgenic lettuce (Lactuca sativa) |
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Authors: | Nguyen-Xuan Huy Young-Sook Kim Sang-Chel Jun Zhewu Jin Seung-Moon Park Moon-Sik Yang Tae-Geum Kim |
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Institution: | (1) Department of Molecular Biology, Chonbuk National University, Jeonju, Chonbuk, 561-756, Republic of Korea;(2) Jeonju Center, Korea Basic Science Institute, Jeonju, 561-756, Republic of Korea |
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Abstract: | Plant-based vaccines have been produced in transgenic plants including tobacco, potatoes, corn, and rice. However, these plants
are not suitable for administration without cooking. To overcome this obstacle, a fusion gene encoding the synthetic enterotoxigenic
Escherichia coli heat-labile enterotoxin B subunit genetically fused with a synthetic neutralizing epitope of porcine epidemic diarrhea virus
(sLTB-sCOE) was introduced into lettuce cells (Lactuca sativa) by Agrobacterium-mediated transformation methods. The integration and expression of the sLTB-sCOE fusion gene was confirmed in transgenic
lettuce by genomic DNA PCR amplification and Northern blot analysis, respectively. Synthesis and assembly of the LTB-COE fusion
protein into oligomeric structures with pentamer size were observed in transgenic plant extracts by Western blot analysis
with anti-LTB or anti-COE antibodies. The binding of plantproduced LTB-COE to intestinal epithelial cell membrane glycolipid
receptors was confirmed by GM1-ganglioside enzyme-linked immunosorbent assay (GM1-ELISA). Based on the ELISA results, LTB-COE fusion protein made up about 0.026∼0.048% of the total soluble protein in the
transgenic lettuce leaf tissues. The synthesis and assembly of LTB-COE monomers into biologically active oligomers in transgenic
lettuce leaf tissues demonstrates the feasibility of using uncooked edible plant-based vaccines for mucosal immunization. |
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