Isolation, Purification And Determination Of Foot-mouth Disease Virus Surface Antigen Fusing Protein CTB-VP1 Expressed In Transgenic Potato

The foot-mouth disease is a kind of zymosis which damage domestic animal with high morbidity, fast outbreak and severe endangerment. The clinical features of FMD are the production of blister and ulcer at oral mucosa, hoof or skin of breast, which would lead to people's panic and remarkable economic losses. The way to produce FMD vaccine by plant bioreactors had become a study hot point, which was safe, sTab.le and low cost to defend and control FMD. A binary vector fusing VP1 and CTB (Cholera toxin subunit B), driven by potato patatin promoter and oriented by the coding region of endoplasmic reticulum transit peptide, was constructed and transformed into potato. Plantlets with antibiotic resistance were screened using PCR. Consequently, transgenic potatoes expressing fusing VP1 and CTB were obtained. In this study, Western blotting verifed that CTB-VP1 was expressed in the transgenic potato, and the techniques for tuber-plantlet rapid reproduction and mini-tuber induction in lab and greenhouse was set up. Isolation, purification and detection of protein CTB-VP1 expressed in transgenic potato offered theory and technical base for mass production of CTB-VP1 from transgenic potato.The specific achievements are as following.1. The result of Western blotting indicated that CTB-VP1 did express in the transgenic potato, the molecular weight of CTB-VP1 is about 52 kD.2. A technique was set up for potato plantlets rapid multiplication and mini-tuber induction. The optimized propagation medium was MS. The optimized condition of of mini-tuber induction was that explants on MS media containing 3 % sucrose was cultured under light for 10 d, then added solution of mini-tuber induction and turned to culture without light, mini-tubers generated after culturing for 20 d, mini-tubers with average weight 3 g/tuber were harvested after plantlets were transplanted in greenhouse for 90 d. Plantlets on the medium with 300 mg/L chlorocholine chloride was stored for 120 d.3. The method for extracting raw protein from mini-tubers was identified, it was as following. Mini-tubers was scraped into bits after kept at - 80 °C overnight, grind and homogenize as fine as possible with a little of quartz, 2-β mercapto ethanol and polyvinylpyrrolidone, adding isolation buffer of three times of minitube fresh weight, then sonicate for 30 min. The optimized isolation buffer was 0.1 M Na₂HPO₄-NaH₂PO₄ The optimized pH of isolation buffer was 7.4. The optimized concentration range of ammonium sulfate to precipitate the raw protein was 30 ～ 70% by SDS-PACE and Western blotting analyses.4. The optimized condition for the positive ion exchange chromatography with CM-Sephadex C-50 column was, in this experiment, 0.02 M phosphoric buffer with pH 6.0 as equilibrium solution, 50 mL loading volume, 0.05 0.30 mol/L NaCl with pH 6.5 for gradient elution, 0.5 mL/min flow velocity during process of chromatography. Collections of the chromatography were concentrated by freeze-drying, which yielded more condensed protein than other approaches of condensation. To further purify concentrated proteins collected from the ion exchange chromatography, gel filtration chromatography on Sephadex G-100 column was utilized. The purified protein from Sephadex G-100 column was tested by Wastern blotting, which showed about 52 kD specific signal formed by combination of FMDV and the protein purified. The result showed that the purified protein is target protein CTB-VP1.5. The protein CTB-VP1 purified was inoculated to lab animals. FMD indirect hemagglutination assay indicated there were specific antibodies generated. Positive level of the antibody reached 1:64.