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dc.contributor.authorLiu, Jianyunen_US
dc.date.accessioned2014-03-14T20:20:58Z
dc.date.available2014-03-14T20:20:58Z
dc.date.issued2006-12-06en_US
dc.identifier.otheretd-12202006-220049en_US
dc.identifier.urihttp://hdl.handle.net/10919/30190
dc.description.abstractInterleukin-12 (IL-12), an important immuno-modulator for cell-mediated immunity, shows significant potential as a vaccine adjuvant and anti-cancer therapeutic. However, its clinical application is limited by lack of an effective bioproduction system and by toxicity associated with systemic administration of IL-12. The goals of this research were to determine whether plants can serve as an effective production system for bioactive IL-12, a complex 70kDa glycoprotein cytokine, and whether the plant lectin RTB can facilitate mucosal delivery of IL-12 to immune responsive sites. Transgenic tobacco plants expressing murine IL-12 were generated and characterized. To ensure stochiometric expression of the two separately encoded, disulfide-linked subunits of IL-12 (p35 and p40), a single-chain form of mouse IL-12 (mIL-12) was utilized. Hairy root cultures, as a fast-growing bioproduction system were developed from high expressers of mIL-12. A purification scheme was developed to purify plant-derived mIL-12 from hairy roots and purified mIL-12 was used to assess IL-12 bioactivity in vitro in mouse splenocytes and in vivo in mouse intranasal vaccination trials. Plant-derived mIL-12 triggered induction of interferon-gamma secretion from mouse splenocytes as well as stimulation of cell proliferation with comparable activities to those observed for the animal-cell-derived mIL-12. Mouse vaccination trials using GFP as the antigen and CT as the adjuvant suggested that plant-derived mIL-12 enhanced Th1 immunity and exhibited similar activity to animal-cell-derived mIL-12 in vivo. Plant-derived IL-12 itself was non-immunogenic suggesting conformational equivalency to endogenous mouse IL-12. Ricin B (RTB), the non-toxic carbohydrate-binding subunit of ricin, directs uptake of ricin into mammalian cells and the intracellular trafficking of ricin A, the catalytic subunit of ricin. RTB's function suggests that it may work as a molecular carrier for effective mucosal delivery of IL-12. To prove this hypothesis, transgenic plants producing RTB:IL-12 fusions were generated and characterized. Our results demonstrated that RTB fused to the carboxyl-terminus of IL-12 maintained full lectin activity and IL-12 bioactivity. RTB fused to the amino-terminus of IL-12 did not show lectin activity due to steric hinderance. Purified IL-12:RTB from transgenic plant tissue was tested in an in vitro mucosal-associated lymphoid tissue (MALT) assay. The results indicate that RTB facilitates the binding of IL-12 to the epithelial cells and presentation of IL-12 to immune responsive cells. In conclusion, my research has shown that transgenic plants are capable of producing valuable bioactive proteins, such as IL-12. Plant-derived mIL-12 exhibited similar activity to animal-cell-derived mIL-12 both in vitro and in vivo. Fusion of IL-12 with the RTB lectin facilitates the delivery of IL-12 to mucosal immune responsive cells and thus may serve as a molecular carrier to enhance IL-12 efficacy and reduce the side-effects associated with systemic administration of IL-12.en_US
dc.publisherVirginia Techen_US
dc.relation.haspartJL_dissertation2.pdfen_US
dc.relation.haspartJL_dissertation1.pdfen_US
dc.rightsI hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to Virginia Tech or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report.en_US
dc.subjectadjuvanten_US
dc.subjectrecombinant expressionen_US
dc.subjectlectinen_US
dc.subjectinterleukin-12en_US
dc.subjectglycoproteinen_US
dc.subjectvaccineen_US
dc.subjectplant bioproduction systemen_US
dc.subjectsingle-chain proteinen_US
dc.subjectprotein purificationen_US
dc.titlePlant-derived Murine IL-12 and Ricin B-Murine IL-12 Fusionsen_US
dc.typeDissertationen_US
dc.contributor.departmentPlant Pathology, Physiology, and Weed Scienceen_US
dc.description.degreePh. D.en_US
thesis.degree.namePh. D.en_US
thesis.degree.leveldoctoralen_US
thesis.degree.grantorVirginia Polytechnic Institute and State Universityen_US
thesis.degree.disciplinePlant Pathology, Physiology, and Weed Scienceen_US
dc.contributor.committeechairCramer, Carole L.en_US
dc.contributor.committeememberHe, Yongqunen_US
dc.contributor.committeememberDolbin-MacNab, Megan L.en_US
dc.contributor.committeememberGogal, Robert M. Jr.en_US
dc.identifier.sourceurlhttp://scholar.lib.vt.edu/theses/available/etd-12202006-220049/en_US
dc.contributor.committeecochairNessler, Craig L.en_US
dc.date.sdate2006-12-20en_US
dc.date.rdate2008-01-26
dc.date.adate2007-01-26en_US


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