The Expression Of Porcine Rotavirus VP7 Protein In Tobacco

In recent years, development and utilization of plant vaccine has become a hot topic, it has gradually recognized by the scientific community because it is safe, effective and low-cost. Porcine rotavirus(PRV) is one of the main pathogenic microorganisms which cause porcine virus diarrhea, it currently is a major virus hazard of our pig industry. VP7 protein is the coat protein of rotavirus(RV) particle surface, it is also the major antigen of RV which can cause the body produce neutralizing antibodies to play a role in immune protection. VP4 and VP6 protein are PRV coat protein and the inner shell protein respectively, they are also important protective antigens. In the prevention of PRV infection, together with the VP7, both of them will stimulate the body’s immune response.in the prevention of RV infection, together with the VP7 will stimulate the body’s immune response. In this study, we have constructed the efficient plant expression vector, in order to establish the genetic transformation system of VP7, VP4 and VP6 protein’s expression in tobacco, to lay foundation for the development of PRV plant vaccine.The main experiment results are as follows:(1) According to the amino acid sequence of PRV VP7, we selected the amino acids51-312 which can lead to neutralizing reaction, used the tobacco preferred codons to optimize,synthesized the target gene sequence. We have added the Ω sequence to the upstream of target gene to improve translation, added M cell targeting peptide- Col sequences to the downstream to enhance immune response, added endoplasmic reticulum ER retention signal sequence to express protein directionally, fused 6 His tag for separation and purification of VP7 protein.(2) We have successfully constructed plant expression vector pBI121-VP7, Transformed it into A.tumefaciens EHA105 by freeze-thaw method, used Agrobacterium-mediated injection infection method to transfer VP7 gene into Nicotiana benthamiana, conducted RT-PCR, SDS-PAGE analysis and used His-Tag antibody to perform Western Blotting,Finally preliminary detected the expression of VP7 protein.(3) We have transformed VP7 gene into Nicotiana tabacum using A.tumefaciens EHA105-mediated leaf disc transformation, through the co-culture, selective differentiationand rooting process, finally obtained two transgenic seedlings, tobacco leaves direct PCR result showed no target gene, it need further examination to verify whether VP7 gene stably transformed.(4) We have synthesized optimized VP4 and VP7 fusion gene VP4-7, VP6 gene of PRV,successfully constructed pBI121-VP4-7, pBI121-VP6 plant expression vectors, and laid foundation for the further expression of VP4-VP7 fused protein、VP6 Protein in tobacco and study of divalent or bivalent plant vaccine.