Expression Of Relevant Pathogenic Protein About Porcine Virus Diarrhea By Botanical System

Porcine virus diarrhea is a common and frequent disease in herd of our country.The main pathogens of this disease are PEDV?PRV and TEGV.Because of its strong,age dependent infectivity and high lethality,it has become a serious threaten to pig enterprises.So it is of significance to research and develop relevant vaccines.Based on the research of expression of porcine rotavirus VP7 protein in tobacco,in the experiment we took use of tobacco transient expression system,tobacco stable expression system and Maize endospern expression system.After the construction of recombinant expression vectors about the relevant pathogenic protein,we translated vectors into plant receptors by agrobacterium tumefaciens method.And then we evaluate the expression level of aimed protein,which if used to offer foundations for further research of plant derived virus vaccine.The main results we obtained are:(1)We choose aa248-aa789 of S protein in PEDV,where the main antigen sites exist.Referring to tobacco preferred codons,we optimized its gene order,and then sent the order to bio corporation for composing.According to different plant receptors,we used 35 S plant promoter and 14 Zein Maize endospern promoter respectively.Finally we structured plant expression vector p BI121-S and Maize endospern expression vector p BI121-p Zein::S.After transferring them into grobacterium tumefaciens by electroporation,We built transgenic plant regeneration system,using agrobacterium tumefaciens method,by which we transferred the S gene into tobacco leaf disc and Maize endospern callus respectively.After selection and defferentiation,we obtained reborn resistant tobacco.(2)Taking wild tobacco as material,we used optimized tobacco transient expression system to transform the VP4-7 protein of PRV,which is a fusion protein of its neutralizing and hemagglutinin antigen.After tobacco infection,we extracted its total protein and purified the protein.Then we analysed the protein before and after purification by SDS-PAGE and Western Blot testing.Taking use of His-tag antibody,we concluded the aimed protein expressed successfully,according to the Westernresult of total protein.But the result of purified protein relevant its size has changed.(3)We choose aa1-aa543 of S protein in TGEV,where the antigen sites exist.we optimized its gene order and added His tag sequence in it.Then we sent the order to bio corporation for composing.Then we structured plant expression vector p BI121-TS successfully,which laid a foundation for further research of plant vaccine.