High-level expression of a human β-site APP cleaving enzyme in transgenic tobacco chloroplasts and its immunogenicity in mice

Abstract

Plastid transformation has to date been applied to the expression of heterologous genes involved in agronomic traits and to the production of useful recombinant proteins. Here, we report a feasibility study for producing the human β-site APP cleaving enzyme (BACE) via transformation of tobacco chloroplasts. Stable integration of human BACE into the plastome was confirmed by PCR. Genomic Southern blot analysis detected the presence of the tobacco aadA and human BACE genes between trnI and trnA in the plastome. Northern blot analysis revealed that the aadA and BACE genes were both properly transcribed into a dicistronic transcriptional unit. Human BACE protein expression in transplastomic tobacco was determined by western blot analysis. ELISA analysis revealed that, based on a dilution series of E. coli-derived BACE as a standard, transplastomic lines accumulated BACE to levels of 2.0% of total soluble proteins. When mice were gavaged with the transplastomic tobacco extracts, they showed an immune response against the BACE antigen. The successful production of plastid-based BACE protein has the potential for developing a plant-based vaccine against Alzheimer disease.

Fig. 1

Introduction of the human BACE gene into the tobacco plastome. a Construction of the transformation vector. The trnI and trnA genes were used as flanking sequences for homologous recombination. The aadA gene conferring spectinomycin resistance was used for selection of transgenic shoots. The aadA and BACE gene were regulated by the rrn promoter (Prrn) and the 3′-UTR of psbA gene (TpsbA) elements. A synthetic ribosome binding site (rbs) was used to achieve translation of BACE. PCR primers are marked as FI and RA with short lines and BglII restriction sites are indicated with the expected size. b Analysis of primary transplastomic plants carrying the human BACE gene integrated into the plastome. Targeted integration of the aadA/BACE expression cassette is indicated by a 3.0 kb product generated from all 8 primary transformed lines; Wt-genomic DNA from non-transformed tobacco plants was used as a negative control

Fig. 2

Analysis of four transplastomic tobacco plants by Southern (a and b) and northern (c) blot. Genomic DNA (5 μg) isolated from non-transformed wild-type plant (Wt) and transplastomic tobacco lines (1–4 correspond to lines B5, C7, E10, and H3) was digested with BglII. DNA blot hybridized with trnA probe (a) and BACE probe (b). c Northern blot analysis of CtVBACE mRNA in transplastomic tobacco plants. Total RNA was extracted from Wt and transplastomic tobacco plants. RNA blot hybridized with BACE probe. Loading was monitored by methylene blue staining of the blot

Fig. 3

Analysis of human BACE protein accumulation in transplastomic plants. a Western blot analysis of TSP from transplastomic tobacco plants and Wt tobacco using anti-BACE serum. Lanes: M, protein weight marker (kDa); P, AB10 (E.coli-produced BACE) cell extracts; Wt, extracts from nontransformed tobacco plant; 1–4, extracts from independent transplastomic tobacco plants B5, C7, E10, and H3, respectively. b BACE protein quantification of transplastomic lines by ELISA analysis. The amount of BACE protein in TSPs (μg antigen mg⁻¹ TSP) was calculated by comparing of absorbance with standard concentrations of E.coli-derived BACE

Fig. 4

a Blood serum anti-BACE antibody response to tobacco plastid-derived BACE in Balb/c mice after oral immunization. Mice were gavaged with extracts from Wt tobacco plants or from the transplastomic tobacco line CtVBACE-B5 with Cholera Toxin as an adjuvant on days 0, 7, and 14. Mice sera were collected before inoculation (days 0) (□); after the third primary immunization (days 14) (▨); and after the booster (day 35) (■). Sera were analyzed by ELISA for induced antibodies specific to BACE. Each bar respresents the average of five immunized mice ± standard error. b Effect of oral immunization of plastid-derived BACE on the body weight in Balb/c mice. The three administrations of plastid-derived BACE are indicated by solid-line arrows, and intraperitoneal booster with 10 μg of yeast-derived recombinant BACE is indicated by a dotted-line arrow. Blood was drawn from the orbital plexus of each mouse on days 0, 14 and 35, to obtain antiserum samples (∇)