Studies On The Fruit-specific Expression Of Cholera Toxin B In Transgenic Tomato Plants

There is an increasing world demand for cheap vaccines that can be readily administered to the population, especially in many undeveloped regions. Since oral immunization could successfully induce specific slgA responses and systemic immune reactions, plant-based vaccine become a very promising strategy because of its special characteristic such as cheap, easy to produce, store and apply. One hurdle before the development of such edible vaccine is the low-expression and diffuse-expression of exogenous gene in recipient plant. So our study is to transform cholera toxin B (CTB) into tomato plant and make it expressed specially in the fruit, providing some considerable base for further experimental of such edible vaccine.Cholera toxin B is a well characterized antigen against cholera, and has been shown to be an effective antigen carrier and adjuvant for coadministered antigens. E8 promoter is fruit-specific promoter of tomatoes. With PCR and enzyme-digestion methods, CTB gene and E8 gene was obtained respectively and then subcloned into binary vector PBI121 with direction. Agrobacterium tumefaciens strain LBA4404 was then induced withPe8-CTB, in order to transfer the target gene into tomato cotyledon.It was found that there are many factors that could affect the regeneration of tomato cotyledon. By orthogonal design of statistics, agents including tomato species, age of cotyledon, concentration of plant hormone and the temperature of cultivation were best assembled, and the frequency of regeneration reached to 98.5%, with average regenerated buds per cytoledon being 6. 8.On the base of the efficient regeneration system, factors that affected transformation such as the Agrobacterium concentration, infiltration time and transform style was optimized. Results showed that best transformation system could be set up by the contidition of 2 days pre-culture, 20-fold-dilution, 10-minute-infiltation of Agrobacterium solution, and 2 days co-cultivation. The frenquency of success transformation reached to 25%, higher than average level at present.Then candidate buds were obtained and elongated. When roots were occurred in the culture medium, the little plants could be removed to outside.Total genome DNA was extracted from the leaves of 26 kanamycin resistant plants. 14 out of 26 were identified to be positive transgenic ones by PCR, dot blot and southern blot.After the ripening of trangenic plants, crude protein was extracted from different tissues and different-period-fruits. CTB protein were determined specially in fruits of 2 transgenicplants by ELISA and Western blot, and reached highest in total mature fruits. The quantity of expression is 455ng and 385ng per fresh weight of fruit, which seemed to be a promising result at present.Animal test is being undertaken, the result is in expectation.Now the fruit-specific transg'enic tomato plants were obtained, with exogenous protein expressed at a considerable level. On such basis, it is no longer impossible to do some animal tests and other further experiments with such plant-based vaccine of CTB.