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Article Title

Detection of major rice allergenic proteins in phytase-transgenic and non-transgenic rice

Abstract

The 14-16 kDa rice allergenic proteins have been reported to be the products of a multigene family, and react most frequently with IgE in serum of rice allergic patients. The aim of this study was to assay the content of endogenous 14-16 kDa rice major allergenic proteins in both phytase-transgenic (GR) and non-transgenic rice (NR) (wild-type, Oryza sative L. cv. Tainung 67). We want to investigate whether the foreign gene will change the allergenicity of rice or not, should be evaluated in order to clarify its pre-market safety. First, the RA17 gene encoding 16 kDa rice allergenic protein was amplified by reverse transcription-polymerase chain reaction (RT-PCR), and cloned into the expression vector pET-29a(+). The constructed expression vector, pET-29a(+)-RA17, was transformed into Escherichia coli BL21 (DE3). The host strain was induced to express recombinant RA17, which was purified by Ni 2+-NTA resin column and used as the antigen for the production of a polyclonal antibody via rabbits. We used the antibody as a detective tool of western blotting and enzyme-linked immunosorbent assay (ELISA) methods. The western blotting analysis showed that the content of major allergenic proteins in phytase-transgenic rice was 96.3% of that in wild-type rice. The content of 14-16 kDa allergens estimated by ELISA were 1.73 ± 0.09 (NR) and 1.66 ± 0.08 mg/g (GR). The results indicated that the level of 14-16 kDa allergens was not significantly different (p > 0.05) between wild-type rice and phytase-transgenic rice, and the transformation of phytase gene into rice plant did not enhance the content of rice major allergenic proteins in transgenic rice.

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