Differential analyses of major allergen proteins in wild-type rice and rice producing a fragment of anti-rotavirus antibody.

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.

• Authors: Yoshikazu Yuki, Shiho Kurokawa, Hiroko Kozuka-Hata, Daisuke Tokuhara, Mio Mejima, Masaharu Kuroda, Masaaki Oyama, Tomoko Nishimaki-Mogami, Reiko Teshima, Hiroshi Kiyono
• Journal: Regulatory toxicology and pharmacology : RTP (Regul Toxicol Pharmacol) Vol. 76 Pg. 128-36 (Apr 2016) ISSN: 1096-0295 [Electronic] Netherlands
• PMID: 26851506 (Publication Type: Journal Article)
• Copyright: Copyright © 2016 Elsevier Inc. All rights reserved.

Chemical References

• Allergens
• Antibodies, Viral
• Antigens, Plant
• Immunoglobulin Fragments
• Immunoglobulin Heavy Chains
• Plant Proteins
• RAG2 protein, Oryza sativa
• Rotavirus Vaccines

Topics

• Allergens (genetics, immunology)
• Antibodies, Viral (biosynthesis, genetics, immunology)
• Antigens, Plant
• Gene Expression Regulation, Plant
• Immunoglobulin Fragments (biosynthesis, genetics, immunology)
• Immunoglobulin Heavy Chains (biosynthesis, genetics, immunology)
• Mass Spectrometry
• Microscopy, Immunoelectron
• Oryza (genetics, immunology, metabolism)
• Plant Proteins (genetics, immunology)
• Plants, Genetically Modified (genetics, immunology, metabolism)
• Proteomics (methods)
• Risk Assessment
• Rotavirus (genetics, immunology)
• Rotavirus Vaccines (biosynthesis, genetics, immunology)
• Two-Dimensional Difference Gel Electrophoresis