Expression Of The Genes Encoding The FMDV Immunogens In Tomato Plants And Arabidopsis Thaliana And Immunization Test Of The Transgenic Tomato Plants For Guinea Pigs Against FMDV Challenge

Foot-and-mouth disease (FMD) is the etiological agent of an important disease of livestock. Vaccination is the major method to prevent FMD. But the conventional inactivated vaccines have many defects, such as the possibility of virus dissemination, and the virulent recovery of vaccine virus. Therefore, it is necessary to develop a safe, efficient and economic FMD vaccine.The use of transgenic plants as vectors for the expression of viral and bacterial antigens has been increasingly tested as alternative methodology for the production of experimental vaccines. Antigens produced in transgenic plants are capable of invoking protective mucosal immune responses against important pathogens as it has already been demonstrated. To explore the feasibility of FMD edible vaccine, in this study, the immunogen gene of VP1 and P12X3C were transferred into the nuclear chromosomal DNA of the tomato plants respectively. In order to evaluate immune response of guinea pigs against FMDV, the extracts of transgenic leaves were injected into guinea pigs. According to the sera antibody levels and the protection of the vaccinated guinea pigs against challenge with 100ID₅₀ FMDV, probed into the immunogenicity of the target protein expressed in transgenic plants.Using plant seeds to produce target proteins could be used as food addition or oral vaccines. In this study, the structural protein VP1 gene was transformed into Arabidopsis thaliana and expression of VP1 in the genome of transgenic Arabidopsis thaliana was performed by molecular biology analysis. The main results presented in this thesis are as follows:1)The plant constitutive-expression vector pBin438/VP1 with VP1 and pBin438/P12X3C with multi-genes of FMDV were constructed respectively, multi-genes P12X3C encode for the FMDV O/China/99 genomic regions P1(1A,1B,1C,1D), 2A, 3C and a part of 2B. The sequence of VP1 contain 660nt including complete VP1, a start codon and microsomal retentional signal sequence SEKDEL. The construction of multi-genes P12X3C also contain 3 018nt including full length of P1, 2A, 3C and a part of 2B, a start codon and microsomal retentional signal sequence SEKDEL. The percent identity of VP1 and P12X3C to the same gene from GenBank of FMDV O/China/99 were 100% and 99.6% respectively.2)The optimal tomato transformation system that was mediated by Agrobacterium fumefaciens GV3101 was established. On the base of the efficient regeneration system, factors that affected transformation such as the Agrobacterium fumefaciens concentration, infiltration time and transform style were optimized. Our protocol of plant transformation system was as follow: from eight to ten days post-germination, cotyledon was infiltrated 3⁵min and co-cultivated for 48h with an overnight grown culture of Agrobaterium Tumefaciens on medium of Murashige and Skoog (MS) adding 1.0mg/L ZT and 0.1mg/L IAA. Cotyledons post-culturation with Agrobacterium. Tumefaciens earring pBin438/VP1 and pBin438/P12X3C were directly placed onto the selective medium containing 50mg/L kanamycin and 500mg/L carbenicillin. After six weeks, kanamycin-resistant shoots were removed from calli, and transferred to the rooting medium. The frenquency of success transformations reached to 18%.