Transgenic Tomato Plant Expressing Cholera Toxin B Protein Specifically in Fruit as Edible Vaccine

JIANG Xiao-ling, HE Zhu-mei, CHEN Qing, PENG Zhi-qiang, QI Yu, YU Shou-yi (Institute of Tropical Military Hygiene of First Military Medical University, Guangzhou 510515; Bio-engineering Center of Life-Science Institute of Sun Yat-sen University, Guangzhou 510275)

Cholera toxin B; Transgenic tomato; E8 promoter; Fruit-specific expression

To make cholera toxin B gene(ctb) expressed in ripening tomato fruit under control of fruit-specific E8 promoter and detect its immunity as an edible vaccine by oral immunization of mice, plasmid pJES 1 containing ctb gene with E8 promoter was induced into Agrobacterium tumefaciens strain LBA4404 and used to transform cotyledons of tomato. Kanamycin-resistant shoot regenerants were selected, elongated, rooted and transferred for fruiting in greenhouse. Transgenic plants were confirmed by PCR and Southern blot analysis. Proteins were extracted from leaf, stem, and fruit tissue of transgenic plants and detected by ELISA and Western blot. Serum and mucosal samples drawn from oral immunized and preimmunized mice were analyzed by ELISA. 14 out of 26 kanamycin-resistant shoot regenerants were confirmed to be transgenic by PCR and Southern blot. ELISA analysis demonstrated that recombinant CTB antigen was expressed in fruits of transgenic plants 9 th and 16 th respectively, and the highest amount of CTB antigen reached 455 and 385 ng·g-1FW in ripening tomato fruits. Oral immunization of mice with ripening fruits of plant 9 th led to the induction of both serum and mucosal CTB specific antibodies. Transgenic tomato plant expressing cholera toxin B protein specifically in fruit as edible vaccine was then obtained successfully.