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Development and evaluation of transgenic rice seeds accumulating a type II-collagen tolerogenic peptide

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Abstract

Type II collagen (CII) in joint cartilage is known to be a major auto-antigen in human rheumatoid arthritis. Several animal model- and clinical-studies on tolerance-based immunotherapy for the arthritis have been conducted by administrating synthetic immunodominant peptides through an oral route. In the present study, to produce a tolerogenic peptide with therapeutic potential in transgenic rice plants, a gene construct producing glutelin fusion protein with tandem four repeats of a CII250–270 peptide (residues 250–270) (GluA-4XCII250–270) containing a human T-cell epitope was introduced with a selection marker, hygromycin phosphotransferase gene (hygromycin-resistance gene) (hph), by co-transformation. Several transgenic plants with high and stable expression of gluA-4XCII 250–270 , but no hph, were selected based on both DNA and protein analyses. The GluA-4XCII250–270 fusion proteins were detected as both precursor and processed forms mainly in a glutelin fraction of rice endosperm protein extracts and in protein-body rich fractions prepared by density gradient ultracentrifugation. The amount of accumulated CII250–270 peptide was immunochemically estimated to be about 1 μg per seed. Feeding DBA/1 mice the transgenic rice seeds (25 μg of the peptide per mouse a day) for 2 weeks showed tendencies lowering and delaying serum specific-IgG2a response against subsequent and repeated intraperitoneal-injection of type II collagen. Taken these together, the CII-immunodominant peptide could effectively be produced and accumulated as a glutelin-fusion protein in the transgenic rice seeds, which might be useful as pharmaceutical materials and functional food for prevention and therapy for anti-CII autoimmune diseases like human rheumatoid arthritis.

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Acknowledgments

This work was supported in part by a grant from the Ministry of Agriculture, Forestry and Fisheries of Japan (Integrated research project for plant, insect and animal using genome technology, IP-2002) and by a Grant-in-Aid for the 21st Century COE Program (14COEA12) from the Ministry of Education, Science, Sports and Culture of Japan. The authors are grateful to Dr. Matsumoto of Nippon Ham Co. Ltd and Dr. Komari of JT Inc. for their kind gifts of protein and plasmid samples, respectively.

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Authors and Affiliations

  1. Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa-ku, Nagoya, Aichi, 464-8601, Japan

    Fujio Hashizume, Shingo Hino, Misako Kakehashi, Tetsuya Okajima, Daita Nadano & Tsukasa Matsuda

  2. Mie Prefecture Agricultural Research Institute, 530 Ureshinokawakita-cho, Matsusaka, Mie, 515-2316, Japan

    Fujio Hashizume

  3. Department of Life Science, Graduate School of Bioresources, Mie University, Tsu, Japan

    Naohito Aoki

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  1. Fujio Hashizume
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  2. Shingo Hino
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  3. Misako Kakehashi
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  4. Tetsuya Okajima
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  5. Daita Nadano
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  7. Tsukasa Matsuda
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Correspondence to Tsukasa Matsuda.

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Hashizume, F., Hino, S., Kakehashi, M. et al. Development and evaluation of transgenic rice seeds accumulating a type II-collagen tolerogenic peptide. Transgenic Res 17, 1117–1129 (2008). https://doi.org/10.1007/s11248-008-9187-2

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  • DOI: https://doi.org/10.1007/s11248-008-9187-2

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