Expression of a multi-epitope DPT fusion protein in transplastomic tobacco plants retains both antigenicity and immunogenicity of all three components of the functional oligomer

Abstract

Expression of genes in plant chloroplasts provides an opportunity for enhanced production of target proteins. We report the introduction and expression of a fusion DPT protein containing immunoprotective exotoxin epitopes of Corynebacterium diphtheriae, Bordetella pertussis, and Clostridium tetani in tobacco chloroplasts. Using biolistic-mediated transformation, a plant-optimized synthetic DPT gene was successfully transferred to tobacco plastomes. Putative transplastomic T0 plants were identified by PCR, and Southern blot analysis confirmed homoplasmy in T1 progeny. ELISA assays demonstrated that the DPT protein retained antigenicity of the three components of the fusion protein. The highest level of expression in these transplastomic plants reached 0.8% of total soluble protein. To assess whether the functional recombinant protein expressed in tobacco plants would induce specific antibodies in test animals, a mice feeding experiment was conducted. For mice orally immunized with freeze-dried transplastomic leaves, production of IgG and IgA antibodies specific to each toxin were detected in serum and mucosal tissues.

Fig. 1

a Schematic diagram of the plastid transformation vector harboring the synthesized DPT gene. This gene is under the control of the Prrn promoter; moreover, the 5′-UTR of phage T7 gene 10 contains a GGAGG ribosome binding site. This gene is inserted at the trnR/trnN insertion site into the tobacco chloroplast genome. RbcL 3, 3′ untranslated region of the rbcL gene; aadA, spectinomycin resistance gene. 1F and 2R correspond to primers used for the detection of the DPT gene; while, 3P and 4T, primers used to detect transplastomic lines. b PCR analysis of plants using 1F and 2R primers for the DPT gene. A specific 0.6-kb PCR band was detected in putative transplastomic tobacco plants (1–4 correspond to lines B3, C1, D3, and E1), but it was absent in wild-type tobacco (Wt). c T0 mature plants transferred to soil and grown in the greenhouse exhibiting normal phenotypes. d T1 seeds germinating on a medium containing 500 mg/L spectinomycin together along with wild-type (Wt). e PCR analysis using primers 3P and 4T; a 2 kb product is amplified from transplastomic plants (1–2 correspond to lines B3 and E1) which is absent in the wild-type (Wt)

Fig. 2

Southern blot analysis of transplastomic T1 lines. Maps of wild-type (a) and transplastomic (b) genomes showing the positions of HindIII and XbaI sites; broken lines indicate the expected size fragments. Southern blot analysis confirmed transgene integration in the plastid genome. DNA was digested by either HindIII (c) or XbaI (d), and blots were hybridized to a probe targeting a fragment of the trnN gene. When DNA was digested with SacI (e), the DPT probe hybridized to a 1.8 kb fragment in the transplastomic plants (1–2 correspond to lines B3 and E1); Wt wild-type plant

Fig. 3

Western blot and ELISA analysis. Anti-tetanus toxin (a) or anti-diphtheria toxin (b) Western blot analysis using 20 μg of TSP from three plants of transplastomic tobacco line E1 (lanes 1–3) and non-transformed wild-type plants (Wt). c ELISA assay: 50 ng of total soluble protein (TSP) from these plants was analyzed using an anti-tetanus toxin polyclonal antibody, an anti-diphtheria toxin polyclonal antibody, and an anti-Bordetella pertussis toxin. Values correspond to means of three replications along with standard deviations. d Levels of DPT protein in leaves of these plants. The amount of DPT was determined using ELISA by comparing the plant sample with a standard curve generated using known amounts (0.5, 1.5, 2.5, and 5 ng) of the tetanus toxoid. Levels of the DPT protein are expressed in μg per 100 mg leaf tissue

Fig. 4

Antibody responses in serum and intestinal fluids. Three weekly doses of transplastomic tobacco plant tissues (50 mg) as well as untransformed wild-type tobacco were administered to male BALB/c mice via the oral route. Individual samples were run in duplicate. Antibody levels were determined by ELISA in serum samples diluted 1:10 or intestinal fluids diluted 1:2 from groups: DPT (+) (immunized with DPT toxoids), T-E1 (transplastomic tobacco E1 line), and Wt (wild-type tobacco). DPT-specific serum IgG (a), mucosal IgA (b), and mucosal IgG (c). Mean A492 values ± SD from each experimental group (n = 5 mice) are shown. TT tetanus toxoid, DT diphtheria toxoid, PT pertussis toxoid

Fig. 5

IgG subclasses of anti-DPT antibodies from serum. IgG1 (a) and IgG2a (b) antibody levels were determined by ELISA in serum diluted 1:10 from the following BALB/c mice: DPT (+) (immunized with DPT toxoids), T-E1 (transplastomic tobacco E1 line), and Wt (wild-type tobacco). Individual samples were run in duplicate. Mean A492 values ± SD from each experimental group (n = 5 mice) are shown. TT tetanus toxoid, DT diphtheria toxoid, PT pertussis toxoid