Development and evaluation of transgenic rice seeds accumulating a type II-collagen tolerogenic peptide.
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 CII(250-270) peptide (residues 250-270) (GluA-4XCII(250-270)) 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-4XCII(250-270) 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 CII(250-270) peptide was immunochemically estimated to be about 1 microg per seed. Feeding DBA/1 mice the transgenic rice seeds (25 microg 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.
Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa-ku, Nagoya, Aichi 464-8601, Japan.
Fujio
F
Hashizume
Shingo
S
Hino
Misako
M
Kakehashi
Tetsuya
T
Okajima
Daita
D
Nadano
Naohito
N
Aoki
Tsukasa
T
Matsuda
D024043:Collagen Type II / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005819:Genetic Markers; D006801:Humans; D007108:Immune Tolerance; D012275:Oryza / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D010455:Peptides / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification / Q000378:metabolism; D030821:Plants, Genetically Modified / Q000276:immunology / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D012639:Seeds / Q000235:genetics / Q000276:immunology* / Q000378:metabolism
D024043:Collagen Type II; D005819:Genetic Markers; D010455:Peptides; D011993:Recombinant Fusion Proteins