RESEARCH Expression of Rabies Glycoprotein and Ricin Toxin B Chain (RGP–RTB) Fusion Protein in Tomato Hairy Roots: A Step Towards Oral Vaccination for Rabies Ankit Singh • Subhi Srivastava • Ankita Chouksey • Bhupendra Singh Panwar • Praveen C. Verma • Sribash Roy • Pradhyumna K. Singh • Gauri Saxena • Rakesh Tuli Ó Springer Science+Business Media New York 2014 Abstract Transgenic hairy roots of Solanum lycopersi- cum 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-spe- cific 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 lg in per gram of tissue. Immunoblot- ting 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 opti- mization 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 prep- aration of improved oral vaccine against rabies. Keywords Hairy roots  Rabies glycoprotein (RGP)  Ricin toxin B chain (RTB)  Oral vaccine Introduction Plants are rapidly emerging as bioreactors which provide promising alternative to molecular farming for safe pro- duction and cost-effective therapeutic proteins [1]. How- ever, transgenic plants are raised on a large scale for manufacturing human and animal vaccines against various viral and bacterial pathogenic agents. Molecular farming of some of the important therapeutic agents is still in initial stages and needs to be studied for their efficacy and sca- lability. One such disease is rabies which causes around 60,000 human deaths worldwide annually [2]. Rabies is acute progressive encephalitis and an oldest known disease of mankind. It is caused by a promiscuous neurotropic virus, Lyssavirus that belongs to family Rhabodoviridae. Electronic supplementary material The online version of this article (doi:10.1007/s12033-014-9829-y) contains supplementary material, which is available to authorized users. A. Singh  S. Srivastava  A. Chouksey  P. C. Verma (&)  S. Roy  P. K. Singh Plant Molecular Biology & Genetic Engineering, National Botanical Research Institute, Council of Scientific and Industrial Research, Rana Pratap Marg, Lucknow 226001, UP, India e-mail: praveencverma@yahoo.com; praveencverma@nbri.res.in B. S. Panwar National Research Centre on Plant Biotechnology, LBS Building, IARI, Pusa Campus, New Delhi 110012, India G. Saxena Department of Botany, University of Lucknow, Lucknow 226001, UP, India R. Tuli Department of Biotechnology, National Agri-Food Biotechnology Institute, C-127, Phase VIII, Industrial Area, SAS Nagar, Mohali 160071, Punjab, India 123 Mol Biotechnol DOI 10.1007/s12033-014-9829-y