Transforming Of Helicobacter Pylori Related Proteins CagA,UreB Genes And CTB Fusion Genes In Tomato

The presence of Helicobacter pylori in human gastric mucosa is now established as the aetiological agent of chronic gastritis and most cases of peptic ulcer and gastric adenocarcinoma world-wide. Today it is a well-recognized pathogen that chronically infects up to 50% of the world's human population and 70% in development countries. It is considered as a class I carcinogen by the World Health Organization. CTB, even in the absence of the toxicity inducing A subunit, also expresses powerful mucosal adjuvant activity. CTB together with antigens increase antigen-specific mucosal IgA responses, administration of CTB as a carrier conjugated to an antigen leads to the induction of local antigen-specific secretory IgA responses. Applications of edible plant vaccines to generate crucial and valuable protein for prophylactic and therapeutic molecular medicine have come of age. It has been demonstrated by many scientists that plants could express recombined proteins in high levels. Tomato is a model plant for transformation and the ideal vector of edible vaccine.In this paper, we constructed fruit specific plant expression vector of CTB-Linker-ureB fusion gene. We transferred vectors p2301-35SCLCN and p2301-35SCLUN into tomato by Agrobacterium tumefaciens GV3101. Finally we obtained regenerated plants, to express the recombined fusion protein CTB and CagA or UreB, which laid the foundation for the research of establishing transgenic tomato as bioreactors to carry microbe antigen and Hp transgenic plant vaccines. The results are showed as follows:(1) The cotyledons and hypocotyls of tomatoes were transformed by Agrobacterium tumefaciens with recombinant. After resistance selection, differentiation and rooting culture, we got transgenic plants. The Km-resistant shoot formation frequency of tomato explants after transformation was 40%⁶0%.(2) PCR and RT-PCR analysis confirmed that the fusion genes have been integrated into the chromosomal DNA of these transgenic plants.(3) Resistant plants were cultured until maturity and took out the seeds (F₁). F₁ seeds were cultured in soild medium preparing for molecular identification. The positivefrequency was > 90%.(4) Construction of fruit specific promoter expression vector, identified vectors couldbe transferred into tomato explants.