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Expression of Rabies Glycoprotein and Ricin Toxin B Chain (RGP–RTB) Fusion Protein in Tomato Hairy Roots: A Step Towards Oral Vaccination for Rabies

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Abstract

Transgenic hairy roots of Solanum lycopersicum were engineered to express a recombinant protein containing a fusion of rabies glycoprotein and ricin toxin B chain (rgp–rtxB) antigen under the control of constitutive CaMV35S promoter. Asialofetuin-mediated direct ELISA of transgenic hairy root extracts was performed using polyclonal anti-rabies antibodies (Ab1) and epitope-specific peptidal anti-RGP (Ab2) antibodies which confirmed the expression of functionally viable RGP–RTB fusion protein. Direct ELISA based on asialofetuin-binding activity was used to screen crude protein extracts from five transgenic hairy root lines. Expressions of RGP–RTB fusion protein in different tomato hairy root lines varied between 1.4 and 8 µg in per gram of tissue. Immunoblotting assay of RGP–RTB fusion protein from these lines showed a protein band on monomeric size of ~84 kDa after denaturation. Tomato hairy root line H03 showed highest level of RGP–RTB protein expression (1.14 %) and was used further in bench-top bioreactor for the optimization of scale-up process to produce large quantity of recombinant protein. Partially purified RGP–RTB fusion protein was able to induce the immune response in BALB/c mice after intra-mucosal immunization. In the present investigation, we have not only successfully scaled up the hairy root culture but also established the utility of this system to produce vaccine antigen which subsequently will reduce the total production cost for implementing rabies vaccination programs in developing nations. This study in a way aims to provide consolidated base for low-cost preparation of improved oral vaccine against rabies.

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Acknowledgments

Authors are thankful to Dr. Shakti Mehrotra (Young Scientist, CSIR-Central Institute of Medicinal & Aromatic Plants, Lucknow, UP) for her kind help in bioreactor related studies.

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Authors and Affiliations

  1. Plant Molecular Biology & Genetic Engineering, National Botanical Research Institute, Council of Scientific and Industrial Research, Rana Pratap Marg, Lucknow, 226001, UP, India

    Ankit Singh, Subhi Srivastava, Ankita Chouksey, Praveen C. Verma, Sribash Roy & Pradhyumna K. Singh

  2. National Research Centre on Plant Biotechnology, LBS Building, IARI, Pusa Campus, New Delhi, 110012, India

    Bhupendra Singh Panwar

  3. Department of Botany, University of Lucknow, Lucknow, 226001, UP, India

    Gauri Saxena

  4. Department of Biotechnology, National Agri-Food Biotechnology Institute, C-127, Phase VIII, Industrial Area, SAS Nagar, Mohali, 160071, Punjab, India

    Rakesh Tuli

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  1. Ankit Singh
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Correspondence to Praveen C. Verma.

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Singh, A., Srivastava, S., Chouksey, A. et al. Expression of Rabies Glycoprotein and Ricin Toxin B Chain (RGP–RTB) Fusion Protein in Tomato Hairy Roots: A Step Towards Oral Vaccination for Rabies. Mol Biotechnol 57, 359–370 (2015). https://doi.org/10.1007/s12033-014-9829-y

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