The Expression Of Recombinant Rotavirus Multi-epitope In Tobacco

In this experiment,the DNA fragment of the epitope from RV VP4 gene was obtained by PCR,at the same time, the DNA fragments of the epitopes from RV VP7 gene , universial helper T lymphocyte epitope and an activated site of tetanus toxin were synthesized, these epitopes were comparted by Glycin and Proline to keep independent. Then these epitopes fragments were linked by over lap extension PCR, enzyme digestion and ligation,and formed a recombinant RV multi-epitope antigen gene(RE),which was inserted into the prokaryotic expression vector pTHio His B. the recombinant pTHio -RE was expressed in E.coli BL21. The relative molecular mass of protein pTHio -RE corresponding to the planed sequence in SDS—PAGE,the molecular mass was 44.7KDa .Then the ELISA experiment was used for checking up the expression of the recombinant protein.The ELISA result indicated that the recombinant RV multi-epitope antigen gene can induce immunoreaction which show blue . The modified multi-epitope gene fragment of rotavirus was insert into the plant expression vector pE1802,then the plant expression vectors pE–RE 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. Transformed shoots were selected on solidified medium Containing Kanamycin. 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 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.