Document Type: Original Article
Department of Agricultural Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran
Introduction: The production and purification cost of a recombinant protein in plants is lower than other conventional systems, such as bacteria. Comparing different parts of the plant, the hairy root is known to be a suitable bioreactor to produce recombinant proteins including vaccine candidate immunogens. Among the most important causes of diarrhea, E. coli and Vibrio cholerae cause the disease by producing a toxin. The B subunit of this toxin is an appropriate candidate for vaccine development because of its non-toxicity and exposure to the immune system.
Materials and Methods: To investigate the efficiency of transgenic hairy roots in the production of recombinant protein, two methods were considered: direct (induction of hairy root with recombinant Agrobacterium rhizogenes) and indirect (production of transgenic plant with Agrobacterium tumefaciens and then polluting it with non-transgenic A. rhizogenes). To compare the 2 methods, GUS protein expression was used as a reporter and chimeric protein LSC (consisting of ltB-stxB-ctxB) as an antigenic protein. Transformation of tobacco hairy root was confirmed by genomic Polymerase chain reaction (PCR). Gene expression comparison was investigated by semi-quantitative ELISA and enzymatic activity.
Results: The results show that the activity of GUS reporter enzyme in the indirect method is seven times more than that of the direct method. Likewise, the expression of LSC recombinant protein in the indirect transformation was 1.5 times more than in direct method.
Conclusions: Comparing of these two methods indicated that the hairy roots in the indirect method yield higher protein content than in the direct method.