Study On The Production Process Of Pig Foot-and-mouth Disease Type O Compound Epitope Protein Vaccine

Foot-and-Mouth Disease(FMD)is a fulminating infectious disease that seriously harms the production of animal husbandry.China's livestock industry and the rural economy were affected greatly after the introduction of type O Burma 98(Mya 98)in February,2010;and the new round Pan Asian(PanAsia-1)system in March,2011.Comprehensive measures of immunization and culling were taken to prevent and control FMD in China,livestocks are inoculated with inactivated vaccine forcibly.,However,because of the weak immunogenicity of FMDV itself and the short duration of the immune system,hidden danger of biological safety still exist,whis is not conducive to the restritction of the purification of FMD,thus it is difficult to carry out the prevention and control work.Therefore,more researchers have paid attention to the invesigation of FMD genetic engineering vaccine.In this study,the production process of type O FMD genetically engineering bacteria constructed in the laboratory was amplified by industrialization,and the production process of a mature and stable type O FMD composite epitope protein vaccine was established by testing the effect evaluation of each process.The morphology,enzyme digestion,expression of target protein and reactivity of the preserved strain were identified.After the qualification,the factors such as induction conditions,culture medium,inoculation quantity,pH and feeding method were optimized to obtain the maximum number of bacteria in the unit of the culture volume.Turbidimetric method was used to measure the optical density of the fermentation broth,and the weight of the bacteria was calculated to analyze the effect of various optimization parameters.The results showed that the optimum incubation time for fermentation was 8h,the induction time and temperature were 8h and 37 ?,respectively.100mmol/L of phosphate was the optimum concentration,with 1mL/L of the trace element solution;the optimum inoculation amount was 5%,pH between 7.0 and 7.2,with 30% of liquid amount;The variable index constant pH mixed flow was added,including ammonia and glycerin as filling solution.Glycerin is more bebeficial than glucose to obtain the target product.According to the Escherichia coli liquid fermented by the upstream process,the preparation scheme of the intermediate products was designed.The hollow fiber system was used to collect the bacteria,and the flux was maintained during the collection process.High pressure homogenizer was used to break the cells under different pressures.The inclusion bodies were purified by affinity chromatography,and eluted with different concentrations of imidazole.The optimum imidazole elution concentration was 100 mM.The purified protein was refolding at different temperatures and dilution rates,and the optimal reactivity conditions were determined by examining the activity and concentration of the refolding protein.Finally,filtration and sterilization were carried out to remove the bacteria.Sterility test was conducted for the samples,the protein concentration was about 1 mg/mL through the BCA method,the protein endotoxin content of was less than 50 EU/mLby the gel method,and the protein activity was 1:1024 by indirect hemagglutination,the expression product was 25.5 Ku by SDS-PAGE analysis,Image Lab software was used to analyze the protein,with purity was over 90%.The results of Western Blot showed that the transfer to the nitrocellulose membrane was reacting with the positive serum of bovine FMDV.According to the existing emulsification technology of the inactivated vaccine of foot and mouth disease,ISA 206 and ISA 201 VG oil adjuvant were used to emulsify the vaccine with 4.6: 5.4,1,1,5.4,4.6 water phase and oil phase ratio,respectively.For 15 months at 2~8?,sampled at third,sixth,ninth,twelfth and fifteenth months for physical properties evaluation..The efficacy of the two adjuvants was compared with that of the emulsified vaccine.The results showed that the best proportion of water phase and oil phase was 1:1.Vaccine formulations,appearance,stability and viscosity all meet the quality requirement of product.The emulsion prepared by two kinds of oil adjuvant can induce the immune pig to produce more than 1:64antibodies,The antibody level of ISA 201 VG adjuvant vaccine was higher than that of ISA 206 oil adjuvant,and all of the ISA 201 VG adjuvant immune dose group received full protection.