Transforming Of Helicobacter Pylori Antigens And Adjuvant Fusion Genes In Tobacco And Tomato

Helicobacter pylori (Hp) is the principal cause of most chronic active gastritis and peptic ulcer disease, and also is closely related with gastric cancer and MALT lymphoma. Current therapies for eradicating H. pylori depend on the use of combined antibiotics. However the high cost, low patient compliance, and increasing of resistant strains make these therapies impractical on a large scale. The use of vaccine will be the best effective way to prevent Hp infection. Cytotoxin-associated protein A (CagA) encoded by the cagA gene, is one of the most studied virulence factors of H. pylori and is a highly immunogenic protein. So it is the principle antigen for anti-Hp vaccine research. Hp produces a large amount of urease, which is essential for the survival and pathogenesis of the bacteria. It has been demonstrated that urease B subunit (UreB) could stimulate body to raise immunogenicity and immunoresponse against challenge of H. pylori. Cholera toxin B subunit (CTB) is non-toxic. It has strong immunogenicity and can enhance the immune efficiency and specialties as mucosal adjuvant. 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. Tobacco is a model plant for transformation and tomato is the ideal vector of edible vaccine.In this paper, we constructed plant expression vectors of CTB-cagA_3.4 , CTB-Linker-ureB and CTB-Linker-cagA₁₀₈ fusion gene, and transferred them into tobacco and tomato by Agrobacterium tumefaciens EHA105 or 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 tobacco and tomato as bioreactors to carry microbe antigen and Hp transgenic plant vaccines. The results are showed as follows:1) The fusion gene was inserted into plant expression vectors pCAMBIA1301, pCAMBIA2301 and pBI121 under the control of CaMV 35S promoter. So we got the vectors named pl3-CC₁N, p23-35SCLUN, p23-35SCLCN and p121-CLUN. PCR analysis, enzyme digest identify and DNA sequence analysis showed the expected results. Then recombinants were transferred into Agrobacterium tumefaciens EHA105 or GV3101.2) Prokaryotic expression vector pET28a-CLU was constructed and transferred into E. coli BL-21. It was induced by IPTG and proteins were determined by SDS-PAGE and western-blot.3) The leaves of tobaccos and the cotyledons and hypocotyls of tomatoes weretransformed by Agrobacterium tumefaciens with recombinant. After resistance selection, differentiation and rooting culture, we got transgenic plants. The Km-resistant shoot formation frequency of tobacco leaves after transformation was 100%, while 40%⁶0% was observed in tomato explants.4) PCR and PCR-Southern analysis confirmed that the fusion genes have been integrated into the chromosomal DNA of these transgenic plants. The western-blot to determine protein expression is still on going.