An Investigation Of The Expression Of The VP1 And Mutiple-epitopes Genes Of FMDV In Plants

Foot-and-mouth disease (FMD), caused by foot-and-mouth disease virus, is a kind of contagious disease that is acute, pyretic and highly tangent and affects domestic cloven-hoofed animals, including cattle, sheep, goats and pigs, as well as more than 70 species of wild animals. In recent years, the frequent outbreaks of FMD-free countries have caused heavy economic losses and became terrorist things to the people in all over the world. The introduction of the skilled FMD vaccine has been extremely successful in reducing the number of disease outbreaks in many parts of the world where the disease is enzootic. However, there are a number of concerns and limitations with its use in emergency control programs, and some of these concerns are being addressed by development of new marker vaccines that is low-cost, safe, effective, and is characterized by high eurytopicity and so easily used and generalized for preventing, controlling and eradicating FMD.In recent years, the research on FMD vaccines and its belongings have being carried through from two aspects. One aspect is the studies on finding antigen epitopes. In some reports about identification of cell epitopes, the experiments of proliferative response of peripheral blood mononuclear cells (PBMC) with synthetic peptides of certain length have made us sure that there are many T-cell epitopes in the structural proteins except VP1 and non-structural proteins, and they have the capacity to induce T helper activity and allow cooperative induction of anti-FMDV antibodies by B cell. The other aspect is looking for a kind of expression system that is low-cost, safe and expediently used to produce FMD vaccine, and transgenic plant edible vaccine becomes highlight since its so many advantages. Our reports focus on two parts, PartⅠis about the possibility of Stylosanthes spp. as a expression system to producing FMD vaccine by the experiment that O type FMDV VP1 gene was expressed in Stylosanthes Guianensis ReyanⅡ. Part Ⅱis on the assay of the expressed product of the fused muti-epitopes genes in the different mode by the potato X virus vector.PartⅠthe research on FMD edible vaccine1. Optimizing gene transformation system of Stylosanthes Guianensis ReyanⅡ. In the experiment methods to increase transformation rate of gene in Stylosanthes guianensis cv. ReyaⅡwere tried by adjusting pre-culture time, Agrobacterium consistence, treating Agrobacterium and explants by acetosyringone, extractive sap from plants and Agroinflitration and disposing co-culture medium by proline and AgNO3.The results showed that transformation rate was enhanced when the explants had been re-cultivated in 2 days, or when Agrobacterium were immersed in the 5 times extractive sap from the papaya plants and 10μmol/L acetosyringone, or when the co-culture medium was treated by 1.5 mg/L proline, or when the explants were dealt with Agroinfiltraction. Adjusting Agrobacterium consistence, extractive sap from Stylosanthes spp. and AgNO3 had no significant effect on transformation rate. On the best condition, there is the more obvious effect than CK by Chi-Square analysis.2. Constructing the expression vector transforming Stylosanthes spp.. The O type FMDV VP1 gene was cloned into the plant intermediate expression vector pBI121 by digesting, ligation and tranformation, and the recombinant of pBIVP1. The recombinant plasmid was introduced in A.tumefaciens LBA4404 by chemic CaCl2 transformation method. The engineering bacterium of A.tumefaciens pBIVP1/LBA- 4404 was obtained by Southern dot-blotting. In optimised conditions, the cotyledon of Stylosanthes spp. was transformed by being mediating by pBIVP1/LBA4404.3. Preparing the Poly-antibody of the fused protein of Ubiquitin-VP1. The Ubiquitin gene was obtained from Arabidopsis thaliana by PCR and sequencing assay. The Ubiquitin gene was fused with the VP1 gene, and sub-cloned into the vector pET30a to constructing the expression vector with 6 His-tag on C-terminal and the Ubiquitin gene on the N-terminal. The expression vector was transformed into the competent E.cloi BL21(DE3) by CaCl2 transformation method. By PCR analysis, the recombinant was identified and engineering bacterium was obtained. The fusion protein was expressed mainly in inclusion body. The fusion protein was purified by 6 His-tag affinity chromatography. The target protein was about 54.6% of the total protein of the bacterium. The New Zealand White rabbit was immunized with purified protein. By double gel diffusion test, sera titers reached highest dilution to 1:64.4. Identifying the transgenic plant with molecular bio-technology. PCR, PCR-Southern blotting and Southern blotting analyses of Stylosanthes spp. DNA confirmed that the FMDV VP1 gene was introduced into the plant genome successfully. RT-PCR and Northern blotting analyses confirmed that the target gene was transcribed. Western blotting analysis confirmed that the FMDV VP1 was expressed in transgenic T1 age with prepared poly -antibody. By indirect ELISA, the concentration of the target protein was 0.05‰～5‰of the total soluble protein.5. Immune response in KM mice by orally immunization. The whole plant materials of the transformed line in which VP1 expression level was identified highest and non-transformed lines were dried at 45℃and pulverized as feedstuff additives. 18 KM mice were divided into three groups, namely, P, N and D. Group P, each mouse was orally immunized each time with 80μg of pure FMDV VP1 protein (emulsified in Freund's Adjuvant with a total volume of 0.4 ml). Group D and N, each mouse was fed each time with the feedstuff granules containing 120 mg hay meal of the transformed line and the non-transformed one, respectively, 2 g starch and some honey. All of these mice were immunized orally on days 0, 15, 30 and 45. Blood was collected from the trail veins of the mice before immunization and 10 days after each immunization for testing the presence of anti-FMDV-specific antibodies by ELISA. ELISA showed that the specific antibodies were elicited, and sera titers reached highest dilution to 1:64.PartⅡThe analysis of immune activity of the expression product of fusion antigen epitope genes.1. Obtaining the antigen epitope genes. The single-chain DNA of 5 T-cell epitope genes (21～35aa of 3A of A type FMDV, 196～210aa of 3C of A or O type FMDV, 49～68 aa of VP2 of O type FMDV, 81～100aa of VP3 of O type FMDV, 20～40aa of VP4 of O type FMDV) and 2 B-cell epitope genes (138～160aa of O type FMDV, 137～160aa of A type FMDV) were synthesized by the company. These single-chain DNA were amplified into double-chain DNA with Klenow enzymes. 2. T-cell antigen genes or B-cell antigen genes were fused respectively. By gene splicing by overlap extension technique, 5 T-cell epitope genes were fused together into T with 4 times PCR, and 2 B-cell epitope genes were fused together into B with 2 times PCR. At the same time, the cloning of O type FMDV VP1 gene was obtained from the plasmid pBIVP1 with the enzyme sites of the expression vector.3. Constructing the intermediate vector with the fusion antigen genes in different assembled method. With AvaⅢand PstⅠwhich can produce uniform stickiness terminal, enzyme-digestion and ligation reactions were done three times and the intermediate vector (pMD-T-B-T, pMD-T-T-B, pMD-B-T-T) were obtained and on these vectors, T and B existed in different position.4. Constructing the plant virus expression vector. Every antigen gene (T, B, T-B-T, T-T-B, B-T-T and VP1 ) were cloned into potato X virus expression vector (PVX), and by PCR analysis of recombinant, the plant virus expression vector (PVX-T, PVX-B,PVX-T-B-T, PVX-T-T-B, PVX-B-T-T, PVX-VP1) were obtained and they were transformed into A. tumefaciens GV3301+PLICSa.5. The tobacco were infected by engineering bacterium of A.tumefaciens, respectively. RT-PCR analysis conformed that all of antigen genes were transcribed.6. The expression products of all of antigen genes were extracted and at the same concentration, ELISA analyses conformed that these products have different antigenicity, and T-B-T was highest, B-T-T take second place, and T-T-B was lowest. But T-T-B was higher than VP1, T or B.