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Expression of anti-K99 scFv in transgenic rice tissues and its functional characterization

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Abstract

As a first step towards manufacturing functional anti-K99 single chain variable antibody fragment (scFv) in a plant system to prevent colibacillosis in neonatal calves, we investigated the feasibility of producing these antibodies in rice plants. Two scFv constructs, with or without the endoplasmic reticulum (ER) targeting KDEL sequence, were introduced into rice for either ER-retention of the recombinant antibody or its secretion. In agreement with several other published reports, extremely low-levels of scFv were produced in rice plants transformed with the construct lacking the ER-targeting sequence. Constructs containing the KDEL sequence resulted in significantly higher levels of the antibody in rice leaves. Although scFv transcripts were found in all three rice tissues analyzed, scFv protein was detected only in the leaf and embryo tissues and not in the endosperm portion of the seed. Functionality of the rice-produced scFv was tested in two in vitro assays, i.e., inhibition of K99-induced horse red blood cell agglutination and inhibition of the attachment of enterotoxigenic Escherichia coli (ETEC) to calf enterocytes. Rice-scFv was found to be functionally equivalent to anti-K99 monoclonal antibody (mAb) in both the assays. The results obtained in this investigation provide valuable information and in combination with other studies of this kind, will be helpful in devising strategies to improve production of useful recombinant proteins in the seeds.

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Acknowledgments

We thank Dr. Umesh Bageshwar (Texas A&M University System Health Science Center) for his valuable suggestions on the Western blotting method. This research was supported by funds from Texas Higher Education Coordinating Board—Advanced Research Program (#999902-0107-1997), Texas AgriLife Research, the Department of Veterinary Pathobiology and TAMU-CONACyT.

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Authors and Affiliations

  1. Institute for Plant Genomics & Biotechnology, Texas A&M University, College Station, TX, 77843-2123, USA

    Ganesan Sunilkumar, LeAnne M. Campbell & Keerti S. Rathore

  2. Department of Veterinary Pathobiology, Texas A&M University, College Station, TX, 77843-4467, USA

    Suryakant D. Waghela

  3. Department of Soil & Crop Sciences, Texas A&M University, College Station, TX, 77843-2474, USA

    Keerti S. Rathore

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  1. Ganesan Sunilkumar
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  2. Suryakant D. Waghela
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  3. LeAnne M. Campbell
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  4. Keerti S. Rathore
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Correspondence to Keerti S. Rathore.

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Sunilkumar, G., Waghela, S.D., Campbell, L.M. et al. Expression of anti-K99 scFv in transgenic rice tissues and its functional characterization. Transgenic Res 18, 347–360 (2009). https://doi.org/10.1007/s11248-008-9223-2

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