The Experssion Of Rotavirus VP4 And VP7 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 present,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 immunogenicity can be effectively expressed if the antigens from infectious bacteria or viruses 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 this study, the VP4 and VP7 genes were amplified and modified by PCR. The kozak sequence was placed upstream of the initiation codons of VP4 and VP7, which was advantageous for the gene expression. And the VP4 gene was mutated in a certain point by PCR.By inserting the modified VP4, VP7 gene of rotavirus into plant expression vector pBI121,pBin438 respectively,2 expression plasmids pBI121-VP4 and pBin438-VP7 were constructed and then transformed to Agrobacterium EHA105 directly. Then pBI121-VP4 and pBin438-VP7 were introduced into tobacco leaf disc. Transformed shoots were selected on solid medium containing Kanamycin. These transformed shoots were checked by PCR, Southern-blot and Western-blot.The Southem-blot indicated that most of transformed shoots had the interested gene integrated into the genome of tobacco, but only few transformed shoots were positive in Western-blot assay.This research provided a new method for new rotavirus vaccine, and presented theorical significance and applicative prospect.