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Characterization of an immunomodulatory Der p 2-FIP-fve fusion protein produced in transformed rice suspension cell culture

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Abstract

Der p 2, a major allergen of Dermatophagoides pteronyssinus mites, is one of the most clinically relevant allergens to allergic patients worldwide. FIP-fve protein (Fve) from the golden needle mushroom (Flammulina velutipes) is an immunomodulatory protein with potential Th1-skewed adjuvant properties. Here, we produced and immunologically evaluated a Der p 2-Fve fusion protein as a potential immunotherapeutic for allergic diseases. Using an inducible expression system in cultured rice suspension cells, the recombinant Der p 2-Fve fusion protein (designated as OsDp2Fve) was expressed in rice cells under the control of an α-amylase gene (αAmy8) promoter and secreted under sucrose starvation. OsDp2Fve was partially purified from the cultured medium. The conformation of Der p 2 in OsDp2Fve remains intact as reflected by its unaltered allergenicity, as assessed by human IgE ELISA and histamine release assays, compared to non-fusion Der p 2 protein. Furthermore, the Fve protein expressed in OsDp2Fve retains its in vitro lymphoproliferative activity but loses its hemagglutination and lymphoagglutination effects compared to the native protein. Notably, in vivo evaluation showed that mice administered with OsDp2Fve possessed an enhanced production of Der p 2-specific IgG antibodies without potentiating the production of Der p 2-specific IgE and Th2 effector cytokines in comparison with mice co-administered with native Fve and Der p 2 proteins. These results suggest that the recombinant Der p 2-Fve fusion protein produced in rice suspension cell cultures has a great potential for allergy immunotherapy.

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Acknowledgments

We thank Ms Lee Mei Liew and Ms Hong Mei Wen for their technical assistance. This work was supported by grants from Academia Sinica of the Republic of China and partly supported by the National Medical Research Council, Singapore (project numbers NMRC/1078/2006 and NMRC/EDG/0038/2008).

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Authors and Affiliations

  1. Institute of Biotechnology, National Cheng Kung University, No.1, University Road, Tainan 701, Taiwan, ROC

    Chin-Fen Su

  2. Department of Paediatrics, Yong Loo Lin School of Medicine, National University of Singapore, 5 Lower Kent Ridge Road, Singapore, Singapore, 119074

    I-Chun Kuo, Chiung-Hui Huang, See Voon Seow & Kaw Yan Chua

  3. Department and Institute of Bioagricultural Sciences, National Chiayi University, Chiayi 600, Taiwan, ROC

    Peng-Wen Chen

  4. Institute of Molecular Biology, Academia Sinica, Nankang, Taipei 115, Taiwan, ROC

    Chin-Fen Su & Su-May Yu

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  1. Chin-Fen Su
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Correspondence to Kaw Yan Chua or Su-May Yu.

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The authors Chin-Fen Su and I-Chun Kuo equally contributed to this work.

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Supplementary material 1 (PDF 53 kb)

11248_2011_9518_MOESM2_ESM.pdf

Supplementary Fig. S1. Expression levels of Der p 2-Fve vary among transgenic rice lines. T1 seeds of Der p 2-Fve transgenic lines were germinated for 5 days in the presence of hygromycin, and the embryos were collected for RNA extraction. Ten μg total RNA was loaded in each lane and subjected to northern blot analysis using Der p 2 cDNA as a probe. NT: non-transformed control(PDF 41 kb)

11248_2011_9518_MOESM3_ESM.pdf

Supplementary Fig. S2. Determination of the copy number of the Der p 2-Fve fusion gene in transgenic rice lines. Genomic DNAs of cell lines 7, 36, 51, and 63 were digested with BglII and subjected to Southern blot analysis using hygromycin gene as a probe. NT: non-transformed control (PDF 23 kb)

11248_2011_9518_MOESM4_ESM.pdf

Supplementary Fig. S3. Cytokine profiles from primary splenocyte cultures. The immunization regimen was performed by subcutaneous injections with Der p 2, Der p 2 + Fve, OsDp2Fve, or Der p 2 + Os medium in phosphate buffered saline in the absence of other adjuvants on days 0, 4, 8, and 14. Mice were sacrificed on day 28, and splenocytes were cultured with 10 μg/ml of Der p 2 for 24 h. The expression of IFN-γ, IL-4, IL-5, and IL-13 transcripts from the primary cultures was determined by relative quantitative RT-PCR with reference to the expression levels of GAPDH. Data are expressed as mean ± sem (n = 6). *: p < 0.05; **: p < 0.005 (PDF 43 kb)

11248_2011_9518_MOESM5_ESM.pdf

Supplementary Fig. S4. Human IgE inhibition ELISA. Six Der p 2-positive human sera were absorbed with serially diluted Der p 2 or Dp2Fve for 16 h and then added to Der p 2-coated wells. Inhibitors were serially diluted from 10 nM to 1 pM (8 dosage points). The percentage of inhibition was calculated as [1-(OD405nm with inhibitor/OD405nm without inhibitor)] × 100 (PDF 29 kb)

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Su, CF., Kuo, IC., Chen, PW. et al. Characterization of an immunomodulatory Der p 2-FIP-fve fusion protein produced in transformed rice suspension cell culture. Transgenic Res 21, 177–192 (2012). https://doi.org/10.1007/s11248-011-9518-6

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  • DOI: https://doi.org/10.1007/s11248-011-9518-6

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