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Expression of recombinant antibody (single chain antibody fragment) in transgenic plant Nicotiana tabacum cv. Xanthi

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Abstract

Plants offer an alternative inexpensive and convenient technology for large scale production of recombinant proteins especially recombinant antibodies (plantibodies). In this paper, we describe the expression of a model single chain antibody fragment (B6scFv) in transgenic tobacco. Four different gene constructs of B6scFv with different target signals for expression in different compartments of a tobacco plant cell with and without endoplasmic reticulum (ER) retention signal were used. Agrobacterium mediated plant transformation of B6scFv gene was performed with tobacco leaf explants and the gene in regenerated plants was detected using histochemical GUS assay and PCR. The expression of B6scFv gene was detected by western blotting and the recombinant protein was purified from putative transgenic tobacco plants using metal affinity chromatography. The expression level of recombinant protein was determined by indirect enzyme-linked immunosorbent assay. The highest accumulation of protein was found up to 3.28 % of the total soluble protein (TSP) in plants expressing B6scFv 1003 targeted to the ER, and subsequently expression of 2.9 % of TSP in plants expressing B6scFv 1004 (with target to apoplast with ER retention signal). In contrast, lower expression of 0.78 and 0.58 % of TSP was found in plants expressing antibody fragment in cytosol and apoplast, without ER retention signal. The described method/system could be used in the future for diverse applications including expression of other recombinant molecules in plants for immunomodulation, obtaining pathogen resistance against plant pathogens, altering metabolic pathways and also for the expression of different antibodies of therapeutic and diagnostic uses.

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Abbreviations

ELISA:

Enzyme-linked immunosorbent assay

ER:

Endoplasmic reticulum

GUS:

Beta-glucuronidase

HIV:

Human immunodeficiency virus

scFv:

Single chain variable fragment

rAb:

Recombinant antibody

RBC:

Red blood cells

TSP:

Total soluble protein

YEM:

Yeast extract mannitol

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Acknowledgments

This work was supported by Department of Biotechnology, Govt. of India. We thanks to Dr. A. Tyagi, Department of Plant Molecular Biology, UDSC, New Delhi for providing Agrobacterium strains, and Central Tobacco Research Institute, Rajamundry, Andra Pradesh (ICAR) for providing tobacco seeds. The authors also thank to Dr. M. K. Saxena and Dr. V. Umapathi for their valuable guidance.

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Authors and Affiliations

  1. Department of Biochemistry, C.B.S.H., G. B. Pant University of Agriculture and Technology, Pantnagar, 263145, Uttarakhand, India

    S. Dobhal & S. Agrawal

  2. Department of Biochemistry, University of Delhi South Campus, New Delhi, 110021, India

    V. K. Chaudhary & A. Singh

  3. Department of Molecular Biology and Genetic Engineering, C.B.S.H., G. B. Pant University of Agriculture and Technology, Pantnagar, 263145, Uttarakhand, India

    D. Pandey & A. Kumar

Authors
  1. S. Dobhal
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  2. V. K. Chaudhary
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  3. A. Singh
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  4. D. Pandey
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  5. A. Kumar
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  6. S. Agrawal
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Correspondence to S. Dobhal.

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Dobhal, S., Chaudhary, V.K., Singh, A. et al. Expression of recombinant antibody (single chain antibody fragment) in transgenic plant Nicotiana tabacum cv. Xanthi. Mol Biol Rep 40, 7027–7037 (2013). https://doi.org/10.1007/s11033-013-2822-x

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  • DOI: https://doi.org/10.1007/s11033-013-2822-x

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