| Foot and mouth disease (FMD), caused by foot-and-mouth disease virus (F MDV), is one of the most febrile, acute, high contagious dual hoofed animal v irus diseases, and has been designed as type A disease by the Office Internatio nal des Epizootie, World Organazation Animal Heath.Seriously, FMD is a rapid spread that causes great economic losses to livestock import and export industy ies, and direct threats human health by food safety, FMD therefor becoming ho t subject among coutries. On the contrary with slaughter extinguish way taked by developed countries when FMD outbreak, our country inoculated animals wi th inactived whole-virus for precaution and treatment, which immunes out high titer antibody and has integral immunogenicity to animals, but increase the ris k of inexhaustive vaccine preparations and the virus escape. Therefore, the dev elopment of a safer, and more effective FMD vaccine becoming a new focus. With the boosts of genetic engineering technology, new vaccines emergenced in recent year included the recombinant subunit vaccines, multi-peptide vaccines, DNA vaccine and recombinant live vector vaccines.For current popular foot and mouth disease virus strains in southern China, design and synthesis two gene fragments:Invasion immunstimulatory sequence+VP1(21-60)+VP1(141-160)+VP1(200-213)+VP4(20-40)+NSP3A(21-35)+the T helper e pitopes and Invasion immunstimulatory sequence+[VP1(21-60)+VP1+(141-160)+VP1(200-213)]3+the T helper epitopes, These two fragments were cloned in pro karyotic expression vector pET32a(+) and double digested by BamHI/HindIII. T he results showed that the fusion expression vector pET32a-multi and pET32a-3re were successfully constructed. The constructed expression vector pET32a-m ulti and pET32a-3re were transformed into the competent cells of the host cell E.coli BL21(DE3). The expressions of the target genes induced by IPTG were analyzed by SDS-PAGE, and results showed that recombinant fusion protein e xpression in the form of inclusion body. SDS-PAGE analysis showed that the relative molecular weights of proteins were measured approximately38KD and 50KD, which are consistent with the theoretical molecular weigh. Recombinant fusion proteins were denatued and refolded with urea, purified by nickel affini ty chromatography. and analyzed by Western-blotting. The results showed that f usion protein fmdv-multi, fmdv-3re have good immunological activity, which lai d the foundation for the immune activity of the recombinant protein.After purification, fmdv-multi and fmdv-3re protein were fully emulsified wit h Freund's adjuvant by equal volume, and injected Balb/c mice by subcutaneou s, making inactivate goods seedlings as the control group.Result of OD Value of Recombinant fusion protein antibody in immunized sera detected by blockin g ELISA,evaluated the immune efficacy of candidate vaccines, The results sho wed that both fmdv-multi and fmdv-3re fusion protein induced the produce of anti-type O FMD antibody in mice and immune effects are similar with the co mmercialization vaccine and fmdv-3re protein was better than fmdv-multi protei n. This study established an foundation for the future development of FMD rec ombinant vaccine.In short, through this study we successfilly designed and builded a multi-epit ope vaccine type O foot-and-mouth disease virus, accomplishing large-scale pro karyotic expression and high-throughput purification of recombinant antigens. In this study, through introducing an important immune-enhancing effect T-helper, the candidate vaccine has stronger immune effectiveness. In the production pr ocess,constantly monitor FMDV and keep abreast of the variation of FMDV ep itope, and timely preparating multi-epitope vaccine.which laid the foundation fo r the development of cheap, efficient multi-epitope vaccine. |
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