Expression Of Multiepitope Antigenic Gene And The Truncated Major Surface Antigenic Gene 1 Of Toxoplasma Gondii In Transgenic Tomato Plants

Biopharming, the production of biological protein in transgenic plant systems, has shown great promise in studies performed recent ten years. Many different candidate vaccines from bacterial and viral sources have been expressed in transgenic plants. Compare to other systems, plants offer some distinct advantages, such as edible plants can be grown locally and can be distributed easily without special training or equipments, the ability to carry out post-translational modifications similar to other higher eukaryotes, transgenic plant vaccine can be delivered orally, by eliminating purification steps, it has also been shown that plants can express multiple transgenes at one time. Moreover, in some experiments, if the plant tissues were simply fed to mice, a mucosal immune response occurred.Toxoplasma gondii is an important food-borne parasite, and the organism may cause congenital infection in humans and other animals. We selected the potential protective antigens the multiepitope antigen gene (MAG) and the truncated major surface antigenic gene (tSAGl) of T. gondii. The tomato fruit specific E8 promoter was amplified from tomato (Zhongshu No.5) genomic DNA. Then, the plant expression vectors were constructed containing MAG and tSAGl under the control of the tomato fruit specific E82.2 promoter, E3 5 S-E81.1 double promoter and E35S promoter respectively. Tomato plants were transformed via Agrobacterium-mediated transformation. Trangenic tomato plants were finally obtained after continued kanamycin-resistant screening and tomato shoot, root inducing. PCR analysis confirmed the presence of MAG in transformed plants. RT-PCR analysis showed the presence of MAG specific transcript in tomatoes fruit. The expression recombinant protein was identified by SDS-PAGE and Western blot. Then the inheritance pattern and stability of transgenic tomato T1 plants was preliminarily analyzed by PCR.This study forms a basis to explore the feasibility of developing a new type of tomato fruit-based edible vaccine against T.gondii.I. Cloning and sequencing of tomato fruit-specific E8 promoter from Lycopersicon esculentum (Zhongshu No.5)Objective In order to prepare for exogenous gene transcription and expression fruit-specific in transgenic tomato, tomato fruit-specific E8 promoter was amplified and cloned and sequenced from Lycopersicon esculentum(Zhongshu No.5).Methods The cotyledons of tomato ( Lycopersicon esculentum Zhongshu No.5) plants were collected to extract tomato genomic DNA. Then the fruit-specific E81.1 and E82.2 promoter DNA of tomato were amplified by PCR, and subcloned into pGEM-T vector. After identification by restriction enzymes, the recombinant T-vectors were submitted to sequence.Results PCR products were of predicted length. Digestion with Xba I and Hindlll/BamH I proved that the recombinant T vectors had the inserts with expected length of the target fragments. Homology analysis indicated that tomato fruit-specific E8 promoter was high conservative. E82.2 promoter of Lycopersicon esculentum Zhongshu No.5 shared 99% high homology with E82.2 promoter of Lycopersicon esculentum cherry. Genbank submission number is AF515784.Conclusion Tomato fruit-specific E8 promoter of Lycopersicon esculentum Zhongshu No.5 was successfully cloned, thus making possible the subsequent research in oral vaccine of transgenic tomato.II. Construction of the plant expression vectors containing MAG of Toxoplasma gondiiObjective Construction of the plant expression vectors containing the multiepitope antigenic gene (MAG) of Toxoplasma gondii under the control of the constitutively expressed cauliflower mosaic virus(CaMV) 35S promoter, E35S-E81.1 double promoter and the tomato fruit-specific E8 promoter.MethodsMAG was subcloned into pBAC55 vector, which carrying a CaMV 35S promoter upstream and a Nos terminator downstream , to construct the intermedia plasmid pB35MG. After identification by restriction enzymes digestion and sequence, the E35S/MAG/NOS3' fragment, was cleaved from pB35MG with HindIII and ligated int...