Construction Two Gene Plant Expression Vector For Fusion Gene Of Rotavirus Multi-epitope RE-LTA2/LTB And Its Expression In Tobacco

Rotavirus (RV) is the major pathogen causing Virus-based diarrhea in infants and young animals of the world, which brings serious damages to economy of the world. Up to the pre- sent,there is no effective medicine for the allopathy, therefore, it is necessary to develop an effective rotavirus vaccine.It is suggested that foreign antigen proteins with pharmaceutical can be effectively expressed if the antigens from infectious bacterial or virus are introduced into plants,which provides a good approach to producing vaccine by transgenic plants.VP4 and VP7 are the major capsule protein and neutralized antigens. Therefore, introducing VP4 and VP7 genes into plant to develop edible plant vaccine of rotavirus would provid potential meaning for society and economy.In the experiment, the recombinant RV multi-epitope antigen gene(RE) replaced A subacute of LT and syncretize A2 of LTA2 formed a recombinant RE-LTA2. The recombinant Ligating with related signal peptide of tobacco formed a middle vector(PMD-PBI121-pr1as-RE-LTA2). A set of plant expression with signal peptide (PR1as-RE-LTA2/Ubquitin–LTB) was formed by enzyme digestion and ligation. The set of plant expression was transformed into Agro bacterium EHA105 directly. By using the leaf-disc co-cultivated method ,multi-epitope gene fragment of rotavirus was transferred into tobacco cell respectively. Integration of these genes into the genome of tobacco plants was confirmed by PCR, then abstracted the recombinant protein of the positive plants confirmed by PCR. and detect the recombinant protein using Dot-ELISA and ELISA. It was shown that most of transformants were integrated into the genome of tobacco,and the recombinant RV multi-epitope antigen gene can be truely expressed in plant. This research provides a new mathod for new rotavirus vaccine, which presents theorical significance and applicative prospect.The recombinant plasmids pBI121-Vp4, pBI121-Vp7 were transferred into Agrobacterium tumefaciens EHA105. By using co-cultivated method, the gene Vp4 and Vp7 were transferred into alfalfa cell. Transferred shoots were selected on solid medium containing Kanamycin. Transgenic alfalfa plants were found containing Vp4 and Vp7 gene by PCR and ELISA. These transgenic alfalfa plants, were confirmed expressing Vp4 and Vp7 recombinant protein through SDS-PAGE. Based on ELISA, results showed that recombinant protein expressed by transgenic alfalfa plants could eliciting immunoresponse.