Construction Of CTB/CS3 Gene Expression Vectors And Transformed Into Cucumber

Vibrio cholerae and Enterotoxigenic Escherichia coli (ETEC) are major pathogens that evoke acute diarrhea among children worldwide and travelers to developing countries. To produce the multicomponent vaccine against diarrhea, a translational fusion with the cholera toxin B subunit (CTB) upstream of CS3, which is one of the prevalent colonization factors (CFs) found in human ETEC strains, was constructed. Because of the capability of GM1 binding, CTB can enhance vaccine efficacy by inducing mucosal and systemic immune responses.Plant bioreactor is a safe and cheap protein production system. However, expression levels of foreign antigens in plant tissues remain quite low. In this study, a series of plant expression vectors were constructed to comparing the expression level of CTB/CS3 in different organs, and transformed by co-cultivating leafdisc with Agrobacterium strains LBA4404 harboring target vector. Transgenic tobacco analysis indicate that the CTB/CS3 gene were incorporated into genomic DNA and expressed CTB/CS3 protein efficiently, and the plant expression vector pBinCTBsp/CSkdel has the highest expression.To use cucumber as a bioreactor producing CTB/CS3 protein against diarrhea, -glucuronidase(GUS) gene was used as a reporter gene to optimize regeneration and transformation system of 649. Cotyledon and cotyledon node are all good explant for cucumber transformation compared to hypocotyledonary axis. MS medium containing 1mg/L NAA and Img/L 6-BA are suitable for transformation of cucumber.The pBinCTBsp/CSkdel gene was introduced into cucumber by Agrobacterium-mediated transformation. After two month of selection, Kanamycin-resistant callus have been formed.