Stable expression of Mtb antigens with CTB in edible cucumber plants (whose fruits are eaten raw) in sufficient amount possibly would facilitate development of a safe, affordable and orally delivered self-adjuvanted, novel dual antigen based subunit vaccine against TB.

A novel combination of two most effective secreted protein antigens specific for Mycobacterium tuberculosis (Mtb), ESAT-6 and MPT-64 (but not present in BCG strains) fused with a cholera toxin B subunit (CTB) and tagged with 6xHis was expressed for the first time in Escherichia coli as well as in transgenic cucumber plants developed using Agrobacterium tumefaciens-mediated transformation. The recombinant fusion protein (His6x.CTB-ESAT6-MPT64) expressed in E. coli was purified by a single-step affinity chromatography and used to produce polyclonal antibodies in rabbit. The transgenic cucumber lines were confirmed by polymerase chain reaction (PCR), Southern blot hybridization, reverse transcriptase PCR (RT-PCR), real-time PCR (qRT-PCR) and expression of recombinant fusion protein by western blot analysis and its quantification by enzyme-linked immunosorbent assay (ELISA). A maximum value of the fusion protein, 478 ng.g-1 (0.030% of the total soluble protein) was obtained in a transgenic cucumber line. Rabbit immunized orally showed a significant increase in serum IgG levels against the fusion protein as compared to the non-immunized rabbit. Conclusions: Stable expression of Mtb antigens with CTB in edible cucumber plants (whose fruits are eaten raw) in sufficient amount possibly would facilitate development of a safe, affordable and orally delivered self-adjuvanted, novel dual antigen based subunit vaccine against TB.