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KMID : 1094720090140060731
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Biotechnology and Bioprocess Engineering
2009 Volume.14 No. 6 p.731 ~ p.737
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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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Huy Nguyen-Xuan
Kim Young-Sook
Jun Sang-Chel
Jin Zhe-Wu
Park Seung-Moon
Yang Moon-Sik
Kim Tae-Geum
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Abstract
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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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KEYWORD
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enterotoxigenic Escherichia coli heat-labile enterotoxin B subunit, edible vaccine, lettuce, mucosal immunization, PEDV
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