Construction of plant expression vectors with fusion gene of Helicobacter pylori cagA，ureB and ctb and its genetic transformation in tobacco

Abstract:

Helicobacter pylori (Hp) is the principal cause of most chronic active gastric and peptic ulcer disease, and also is closely related with gastric cancer and MALT lymphoma. Current vaccines are expensive to produce and deliver, however, transgenic plants expressing recombinant vaccine immunogens offer an attractive and potential inexpensive alternative to vaccination and injection. In this study, plant expression vectors which harbor Hp related proteins CagA and UreB were constructed. Fusion gene ctb-linker-cagA and ctb-linker-ureB were cut from vectors p1300-WxCLCN and p1300-WxCLUN, and then constructed into vector pCAMBIA2301 which was under the control of the CaMV 35S promoter by series molecular methods. Those reconstructed vectors were named p2301-35SCLCN and p2301-35SCLUN and were introduced into Agrobacterium tumefaciens strain EHA105.Tobacco was transformed by co-cultivating leaf discs with Agrobacterium strains harboring fusion genes. The regenerated Kanamycin-resistant transforms were selected, elongated, rooted and transferred for flowering in greenhouse. Recombinant plant expression vectors were confirmed by digestion and PCR and transgenic plants were analyzed by PCR, GUS histochemical assays, PCR-Southern blot. The results show that more than 80% transgenic plants are confirmed to be positive ones and these results also indicate that ctb-linker-cagA and ctb-linker-ureB are integrated into the genomic DNA of the tobacco which laid a solid foundation for the research of establishing transgenic plants as bioreactors to carry microbe antigen and Hp transgenic plant vaccines.