Expression And Immunoactivity Of HBsAg (hepatitis B Virus Surface Antigen) And HEV-NE2 (hepatitis E Virus ORF2 Gene) In Transgenic Tomato

By using cotyledons and hypocotyls of tomatos as transformatic explants, the factors affecting gene transfer mediated by Agrobacterium tumefaciens were systematically examined according to Gus expressing efficiency, and the optimized genetic transformation system of tomato was established. The plant binary expression vector p!301HBs, pBOOHBs carrying hepatitis B virus surface antigen(HBsAg) gene, p!301NE2, p!300NE2 carrying NE2 gene, a fragment of ORF2 region of hepatitis E virus (HEV-NE2), were constructed and introduced into tomato, and hygromycin-resistant plantlets were obtained. The results of PCR and Southern blotting of tomato genomic DNA demonstrated that the target gene was integrated into the genome of tomato plants. The examination of ELISA suggested the HBsAg gene has expressed correctly in transgenic tomato plants. The immunization experiment indicated that the HBsAg-transgenic plant derived vaccine can produce booster immune responses in Balb/c mice primed with commercial vaccine. All these demonstrated the perfect perspective of plant-derived edible vaccine and provided experimental basis and guiding theory for the practicality of plant-derived edible vaccine.1.Construction of plant binary expression vector: The HBsAg gene linked with CaMV35S promoter, nos terminal and Q enhancer was inserted into the mediate expression vectors pCAMBIA1301 (containing hygromycin- resistant gene, kanamycin-resistant gene, Gus gene) and pCAMB!A1300 (containing Hyg- resistant gene, Kan-resistant gene, no Gus gene), respectively, to form the reconstructed plant binary expression plasmids p!301HBs and p!300HBs. In the same way, the plant binary expression plasmids p!301NE2, p!300NE2 carrying HEV-NE2 gene were constructed. Restriction endonuclease analysis indicated that these plasmids were directly introduced into strains LBA4404, EHA105, AGL1.2. Genetic transformation system establishment: Genetic transformation conditions were examined by Gus expression, and an efficient genetic transformation system was developed. That is: 9-days-old explants pre-cultured for 3 days in darkness or 12-days-old explants pre-cultured for 2 days in darkness were co-cultured with EH A105 for 3 days in darkness, then selected by 20mg/g hygromycin. 500mg/g carbenicillin(carb) which will be replaced by cefotaxine(cef) later was used as inhibitor of the Agrobacterium tumefaciens, both of which will be reduced gradually.Using the above-mentioned transformation system, the p!300HBs without Gus gene, and the HEV-NE2 gene were introduced into tomato, many hyg-resistent plantlets were acquired too. This demonstrated that the established transformation system is reliable, repeatable, thus efficient.3. Distinguishing the hyg-resistant plantlets and determining expression amount of target protein: The integration of foreign DNA into transgenic tomato was confirmed by hygromycin resistance, Gus assay, PCR and Southern blotting. The immunoacticity of recombinant HBsAg, HEV-NE2 was shown by ELISA. The amount of HBsAg in transgenic tomato is about 240-300ng/g fresh weight, and 0.003 percent of total soluble protein, whereas the amount of HEV-NE2 is very low. Up to now, we have obtained about 40 HBsAg-transgenic plants bearing more than 100 tomato fruits, 7 NE2~transgenic plants, one of which has born 2 fruits.4. Immunogenicity of transgenic plant-derived HBsAg: Mice primed with injection of a sub-immunogenic dose(0.5 u g/g) of commercial vaccine, were fed with transgenic tissue or injected with purified extraction. Four weeks later, the mice boosted special serum antibody of HBsAg.