Change in subcellular localization of overexpressed vaccine peptide in rice endosperm cell that is caused by suppression of endogenous seed storage proteins
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Rice-based peptide vaccine based on T cell epitopes acts as an ideal oral tolerogen for the treatment of type 1 allergic diseases. To improve production yields of oral tolerogen against Japanese cedar pollen allergy, hybrid peptide comprising seven predominant human T cell epitopes (7Crp) derived from Japanese cedar pollen allergens, Cry j 1 and Cry j 2, was produced in transgenic rice seed by expression of its codon optimized gene under the control of the endosperm-specific 26 kD globulin (Glb-1) promoter containing its signal peptide and the simultaneous suppression of endogenous seed storage proteins (SSPs) by RNA interference. Accumulation level of 7Crp peptide produced as a secretory protein was remarkably enhanced by suppression of both the 13–14 kDa prolamins and GluA and GluB glutelins as compared to those under suppression of either of them or in wild type rice. When these SSPs were down-regulated, the 7Crp peptide was observed to be localized in ER lumen as well as ER derived PBs (PB-Is). Especially, accumulation as self-aggregates in ER lumen increased by reduction of the endogenous 13–14 kDa prolamins. It is interesting to note that the absence of C terminal KDEL ER retention signal from the 7Crp peptide resulted in higher level accumulation (116 µg/grain) than that containing the KDEL.
KeywordsGlb-1 promoter Peptide vaccine Rice RNA interference (RNAi) Seed storage proteins
Gut-associated lymphoid tissue
26 kDa Globulin
Protein disulfide isomerase-like
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis
Seed storage protein
We thank Ms. K. Miyashita, Y. Ikemoto and Y. Yajima for technical assistance, Dr. S. Saito (Jikei University of School of Medicine) for providing anti-7Crp monoclonal antibody, and Dr. Kenjiro Ozawa for encourage of this research. M.E was supported by Ministry of Science, Research and Technology of Iran.
FT designed this research. ME, FT and YW conducted experiments. FT and ME wrote this manuscript. All authors read and approved the manuscript.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
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