Institution: | 1. U. S. Department of Agriculture, Agricultural Research Service, Pacific West Area, Western Regional Research Center, 800 Buchanan Street, Albany, CA 94710, USA;2. School of Food Science and Technology, Jiangnan University, 214122 Wuxi, China;3. College of Chemistry and Environmental Engineering, Shenzhen University, Shenzhen 518060, China;4. Division of Pediatric Immunology, Allergy, and Rheumatology, Department of Pediatrics, Stanford University School of Medicine, 269 Campus Drive, Stanford, CA 94305, USA |
Abstract: | To develop oral antibody therapy against rotavirus infection, we previously produced a recombinant fragment of llama heavy-chain antibody to rotavirus (ARP1) in rice seeds (MucoRice-ARP1). We intend to use a purification-free rice powder for clinical application but needed to check whether MucoRice-ARP1 had increased levels of known allergen proteins. For this purpose, we used two-dimensional fluorescence difference gel electrophoresis to compare the allergen protein levels in MucoRice-ARP1 and wild-type rice. We detected no notable differences, except in the levels of α-amylase/trypsin inhibitor–like family proteins. Because by this approach we could not completely separate ARP1 from the proteins of this family, we confirmed the absence of changes in the levels of these allergens by using shotgun mass spectrometry as well as immunoblot. By using immunoelectron microscopy, we also showed that RAG2, a member of the α-amylase/trypsin inhibitor–like protein family, was relocated from protein bodies II to the plasma membrane or cell wall in MucoRice-ARP1 seed. The relocation did not affect the level of RAG2. We demonstrated that most of the known rice allergens were not considerably upregulated by the genetic modification in MucoRice-ARP1. Our data suggest that MucoRice-ARP1 is a potentially safe oral antibody for clinical application. |