Expression Of Classical Swine Fever Virus E2 Protein In Escherichia Coli And Analysis Of Its Immunogenicity

Classical swine fever(CSF)has been classified by the international veterinary organization OIE as class A infectious disease caused by classical swine fever virus(CSFV).In recent years,pigs that died of CSFV infection in China accounted for more than one-third of all dead pigs caused by the epidemic,causing huge economic losses and a waste of resources to the domestic pig industry.China usually adopts vaccinated swine fever attenuated vaccines and other control measures to control the outbreak of swine fever.The epidemic of swine fever has gradually changed from a large-scale epidemic to an irregular epidemic.Although the dose and frequency of vaccine immunization have increased,the spread of swine fever has not been effectively controlled.As a result of the misuse of the attenuated swine fever vaccine and the mutation of the wild strain,chronic and latent infections of swine fever epidemic occurred,making it difficult to allow differentiating infected from vaccinated animals(DIVA)and the epidemic more difficult to control.Therefore,the development of a novel genetic engineering subunit vaccine that can be differentially diagnosed,safe,and highly efficient is an urgent need for the pig industry worldwide.The aim of this study is to prepare an immunogenic CSFV E2 protein which can be expressed in the soluble fraction in E.coli,and then carry out the purification and immunogenicity analysis.The E2 gene was inserted into prokaryotic vector p ET-28 a to construct a recombinant plasmid p ET-28a-E2.The plasmid was transformed into E.coli BL21(DE3)and induced with IPTG.The results of SDS-PAGE electrophoresis and Western Blot proved that E2 protein was successfully expressed.The final concentrations of IPTG,the induction temperature and the induction time were optimized.The results showed that when the cells was induced with 0.1 mmol/L IPTG at 18 °C for 16 h,the highest expression level of E2 protein was achieved.The E2 protein was purified in one step by size exclusion chromatography with a final mass concentration of 0.5 g/L.Western Blot and ELISA results showed that the purified E2 protein could be specifically recognized by the swine fever positive serum under both denatured and non-denatured conditions,indicating that the purified E2 protein was successfully expressed and the activity was good.The purified E2 protein was emulsified with Freund's adjuvant,gel 01 adjuvant or nanomaterials to immunize BALB/C mice.The serum of the mice was collected weekly to determine the antibody titer,the law of antibody ablation and the neutralizing antibody test.The results showed that the antibody levels in the serum of mice continuously increased after immunization and reached the highest level at 56 days,indicating that the E2 protein was immunogenic.In summary,the soluble E2 protein was expressed successfully using the E.coli BL21(DE3)strain,and the purified E2 protein had good immunogenicity.