|Pegg, Sue Ellen|
Submitted to: Molecular Breeding
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/28/2002
Publication Date: 6/28/2002
Citation: CHIKWAMBA, R., MCMURRAY, J., SHOU, H., FRAME, B., PEGG, S., SCOTT, M.P., MASON, H., WANG, K. EXPRESSION OF A SYNTHETIC E. COLI HEAT LABILE ENTEROTOXIN B SUBUNIT (LTB) IN MAIZE. MOLECULAR BREEDING. 2002. v.10. p. 253-265. Interpretive Summary: Edible vaccines are important in human health and veterinary medicine. Biotechnology offers the potential to produce these vaccines in the edible organs of food and feed plants. There are many advantages with this approach. The expense and technology required for vaccine production can be greatly reduced because purification of the vaccine is not necessary. Also, vaccines contained in plant organs may be stored and shipped inexpensively. The E. coli labile toxin B (LT-B) subunit is considered a good carrier for edible vaccines. In native labile toxin, the toxin activity is contained in the A subunit, while the B subunit of this protein delivers the A subunit to it's target cells. Thus, the B subunit of this protein survives digestion and interacts specifically with cells involved in mediating the immune response. Several researchers have designed edible vaccines by fusing vaccines to LT-B. In this manuscript, we describe methods for the production of LT-B in transgenic maize. We designed four different genes for the production of LT-B and compared plants containing these genes on the basis of their accumulation of functional LT-B. Plants producing LT-B specifically in kernels tended to contain higher levels of LT-B in kernels than plants producing LT-B in kernels as well as in other plant tissues. Scientists working with edible vaccines will benefit from this work because they will be able to use the information presented here to design edible vaccines for production in maize.
Technical Abstract: We have produced the B sub-unit of the enterotoxigenic Escherischia coli (ETEC) heat labile enterotoxin (LT-B) in transgenic maize seed. LT-B is a model antigen that induces a strong immune response upon oral administration and enhances immune responses to conjugated and co-administered antigens. Using a synthetic LT-B gene with optimized codon sequence, we examined the role of promoters and the SEKDEL endoplasmic reticulum retention motif in LT-B accumulation in callus and in kernels. Two promoters, the constitutive CaMV 35S promoter and the maize 27 kDa gamma zein promoter, which directs endosperm specific gene expression in maize kernels, regulated LT-B expression. Ganglioside-dependent ELISA analysis showed that using the constitutive promoter, maximum LT-B level detected in callus was 0.04% LT-B in total aqueous extractable protein (TAEP) and 0.01% in R1 kernels of transgenic plants. Using the gamma zein promoter, LT-B accumulation reached 0.07% in R1 kernels. While the SEKDEL motif did not substantially enhance constitutive expression of LT-B from the CaMV 35S promoter, it resulted in increased LT-B levels when combined with the gamma zein promoter. We monitored LT-B levels under greenhouse and field conditions over three generations. Significant variability in gene expression was observed between transgenic events, and between plants within the same event. A maximum of 0.3% LT-B in TAEP was measured in R3 seed of a transgenic line carrying CaMV 35S promoter/LT-B construct. In R3 seed of a transgenic line carrying the gamma zein promoter/LT-B construct, up to 3.7% LT-B in TAEP could be detected. This study demonstrates that maize seed can be used as a production system for functional antigens.