SummaryPeanut allergy is one of the most life-threatening food allergies and one of the serious challenges facing the peanut and food industries. Current proposed solutions focus primarily on ways to alter the immune system of patients allergic to peanut. However, with the advent of genetic engineering novel strategies can be proposed to solve the problem of peanut allergy from the source. The objectives of this study were to eliminate the immunodominant Ara h 2 protein from transgenic peanut using RNA interference (RNAi), and to evaluate the allergenicity of resulting transgenic peanut seeds. A 265-bp-long PCR product was generated from the coding region of Ara h 2 genomic DNA, and cloned as inverted repeats in pHANNIBAL, an RNAi-inducing plant transformation vector. The Ara h 2-specific RNAi transformation cassette was subcloned into a binary pART27 vector to construct plasmid pDK28. Transgenic peanuts were produced by infecting peanut hypocotyl explants with Agrobacterium tumefaciens EHA 105 harbouring the pDK28 construct.A total of 59 kanamycin-resistant peanut plants were regenerated with phenotype and growth rates comparable to wild type. PCR and Southern analyses revealed that 44% of plants stably integrated the transgene. Sandwich ELISA performed using Ara h 2-mAbs revealed a significant ( P < 0.05) reduction in Ara h 2 content in several transgenic seeds.Western immunobloting performed with Ara h 2-mAb corroborated the results obtained with ELISA and showed absence of the Ara h 2 protein from crude extracts of several transgenic seeds of the T 0 plants. The allergenicity of transgenic peanut seeds expressed as IgE binding capacity was evaluated by ELISA using sera of patients allergic to peanut.The data showed a significant decrease in the IgE binding capacity of selected transgenic seeds compared to wild type, hence, demonstrating the feasibility of alleviating peanut allergy using the RNAi technology.

Pulsed ultraviolet light (PUV), a non-thermal food processing technology, is reported to be able to inactivate enzymes and reduce allergen levels from peanut extracts. The objective of this study was to determine if PUV would reduce the allergen levels and allergenic potency of soy extracts. Soy extracts were treated with PUV at various times (2, 4 and 6 min), centrifuged, and analyzed by SDS-PAGE and an indirect ELISA for IgE binding or allergenic potency. Results showed that PUV treatment led to an increase in sample temperature/weight loss but a decrease in the levels of soy allergens (i.e., glycinin and ?-conglycinin) as shown in SDS-PAGE. Allergens were reduced probably through aggregation which increased with treatment time. IgE binding was reduced as well in the following order: 20%, 44% and 50% reductions in absorbance values at 2, 4, 6 min, respectively (the latter two were not significantly different (p < 0.05%) from each other). It was concluded that PUV was capable of reducing the allergenic potency of soy extracts, and that the optimal PUV treatment time was 4 min. Clinical data is still needed before PUV can find an application in the development of less allergenic soybean beverages and products.

Peanut allergy is an IgE-mediated hypersensitivity reaction with an increasing prevalence worldwide. Despite its seriousness, to date, there is no cure. Genetic engineering strategies can provide a solution. The post-transcriptional gene silencing (PTGS) model can be used effectively to knock out the production of allergenic proteins in peanut by specific degradation of the endogenous target messenger RNA (mRNA). Ara h 2, the most potent peanut allergenic protein, was selected as a model to demonstrate the feasibility of this concept. Transgenic peanut plants were produced via microprojectile-mediated transformation of peanut embryos using a plasmid construct, which contains a fragment of the coding region of Ara h 2 linked to an enhanced CaMV 35S constitutive promoter. Molecular analyses, including polymerase chain reaction and Southern blots, confirmed the presence of the stable integration of the Ara h 2 transgene into the peanut genome. Northern hybridization showed the expression of the Ara h 2 transgene in all vegetative tissues of the mature transgenic peanut plants, indicating the stable expression of the truncated Ara h 2 transgene throughout the development of the plants. It is, therefore, reasonable to expect that the truncated Ara h 2 transgene transcripts will be synthesized in the seeds and will trigger the specific degradation of endogenous Ara h 2 mRNA. The next step will be to grow the transgenic peanut plants to full maturity for seed production and to determine the level of allergen Ara h 2.

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