Degree Type

Dissertation

Date of Award

2002

Degree Name

Doctor of Philosophy

Department

Agronomy

Major

Genetics

First Advisor

Kan Wang

Second Advisor

Michael Lee

Abstract

Plants are becoming increasingly important as a production system for biopharmaceuticals and industrially important proteins. The work presented in this dissertation showed that maize can be used as a source and delivery vehicle for oral vaccines. Antigenic proteins from two economically important pathogens, enterotoxigenic Escherichia coli (E. coli ) and the swine transmissible gastroenteritis virus (TGEV) were expressed in transgenic maize.;This study showed that subunits of the E. coli heat labile enterotoxin (LT) can be synthesized in transgenic maize tissues, correctly processed and assembled in maize tissue. The role of regulatory sequences such as promoters, targeting and retention signals in accumulation of LT-B in transgenic maize kernels was studied. The seed specific 27 kDa gamma zein promoter achieved a significantly higher level of LT-B expression in kernels compared to the constitutive CaMV 35S promoter. The use of the endoplasmic reticulum retention motif SEKDEL significantly enhanced kernel accumulation of LT-B. The LT-13 gene was normally transmitted over three generations.;Maize generated LT-B had biochemical, biophysical, and immunogenic properties of the bacterial protein. Oral administration of transgenic maize expressing LT-B in BALB/c mice induced elevated titers of serum and mucosal antibodies, which protected the immunized animals from subsequent challenge with LT and Cholera toxin (CT).;Using two synthetic genes for the LT toxin subunits, LT-A and LT-B, a non-toxic derivative of the heat labile toxin, LTK63, was expressed in transgenic maize callus. This mutant toxin assembled in maize callus tissue, showing that complex folding of foreign antigens could be achieved in transgenic maize tissues. This mutant derivative was shown to be more immunogenic than the bacteria derived LT-B.;We fused an N-terminal domain of the spike (S) protein of the swine transmissible gastroenteritis virus to the A subunit of LT, and coexpressed this fusion with LT-B in transgenic maize callus. Expression of the fusion proteins and LT-B was observed in callus.;This work demonstrates that maize, a key ingredient in food and feed industry, can be used as a source and delivery vehicle of functional antigens for use as oral vaccines. Maize holds great potential for the generation of human and livestock vaccines, and this work lays the foundation for the development of vaccines against other pathogens in transgenic maize.

DOI

https://doi.org/10.31274/rtd-180813-926

Publisher

Digital Repository @ Iowa State University, http://lib.dr.iastate.edu

Copyright Owner

Rachel Kerina Chikwamba

Language

en

Proquest ID

AAI3061819

File Format

application/pdf

File Size

209 pages

Share

COinS