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MucoRice-cholera Toxin B‑subunit, a Rice-based Oral Cholera Vaccine, Down-regulates the Expression of α‑Amylase/trypsin Inhibitor-like Protein Family as Major Rice Allergens

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posted on 05.07.2013, 00:00 by Shiho Kurokawa, Rika Nakamura, Mio Mejima, Hiroko Kozuka-Hata, Masaharu Kuroda, Natsumi Takeyama, Masaaki Oyama, Shigeru Satoh, Hiroshi Kiyono, Takehiro Masumura, Reiko Teshima, Yoshikazu Yuki
To develop a cold chain- and needle/syringe-free rice-based cholera vaccine (MucoRice-CTB) for human use, we previously advanced the MucoRice system by introducing antisense genes specific for endogenous rice storage proteins and produced a molecularly uniform, human-applicable, high-yield MucoRice-CTB devoid of plant-associated sugar. To maintain the cold chain-free property of this vaccine for clinical application, we wanted to use a polished rice powder preparation of MucoRice-CTB without further purification but wondered whether this might cause an unexpected increase in rice allergen protein expression levels in MucoRice-CTB and prompt safety concerns. Therefore, we used two-dimensional fluorescence difference gel electrophoresis and shotgun MS/MS proteomics to compare rice allergen protein expression levels in MucoRice-CTB and wild-type (WT) rice. Both proteomics analyses showed that the only notable change in the expression levels of rice allergen protein in MucoRice-CTB, compared with those in WT rice, was a decrease in the expression levels of α-amylase/trypsin inhibitor-like protein family such as the seed allergen protein RAG2. Real-time PCR analysis showed mRNA of RAG2 reduced in MucoRice-CTB seed. These results demonstrate that no known rice allergens appear to be up-reregulated by genetic modification of MucoRice-CTB, suggesting that MucoRice-CTB has potential as a safe oral cholera vaccine for clinical application.