Preparation Of The Oral Vaccine Loaded With E2 Protein Of Classical Swine Fever Virus And Mucosal Adjuvant Lt Protein And Evaluation On Its Immunization In Mice

Classical swine fever(CSF)is one of the biggest virus infectious disease in pigs.It brings great harm to the pig industry.Despite the existing CSF vaccine plays a good immune effect,but the injection needs a lot of manpower and material resources,which not only increases the stress of pigs,but also increases the cost of pig industry.Therefore,with the development of new vaccines,a convenient and safe way of immunity causes more and more attention.Oral immunization,as a hotspot of vaccination way,has more advantages,such as easy to mass immunization,save manpower and material resources and small animal stress.In this study,oral microsphere vaccine against CSF was prepared based on the liquid gel transition theory,using spray forming,Ca2+ solution,stirring,collection of freeze-drying and other technological means.The E2 protein of classical swine fever virus(CSFV)and mucosal adjuvant LT protein were used as core material,sodium alginate with good biocompatibility was used as the wall material,coated with chitosan outside.The encapsulation efficiency,particle size,different pH solution in the expansion rate and the release quantity and storage stability were tested.In order to find the optimal conditions for the microsphere preparation,the orthogonal analysis of four factors in three levels had been taken.The results showed that the optimal conditions in microsphere preparation were as follows:2.0%sodium alginate(SA),18mL/min spray speed,0.5%CaCl2,and 1400rpm of stirring speed.By using this method,microsphere encapsulation rate was 81.67%,the drug loading was 0.045mg/mg microsphere,and in vitro release rate of the microsphere was minimal in acid environment.The microsphere have good safety performance and high storage stability.To evaluate the immune effect of the oral microsphere vaccine,mice were orally immunized.Enzyme-linked immunosorbent assay(ELISA)method was used for analysis of serum antibodies,nasal wash fluid antibodies and cytokine expression levels.The result showed that after the third immunization,compared to control group,in E2 injection group,E2 microsphere group,E2-LT microsphere groupand LT microsphere group,the specific IgG levels against E2 were significantly increased(P<0.05),and the sIgA levels in E2-LT group and E2 group were significantly increased(P<0.05).As for cytokines,compared with other groups,IFN-γ expression levels in E2 injection group after immunization were highest,while IL-4 expression was highest in E2-LT microsphere group.The expression levels of IL-4 upregulation were earlier than IFN-γ in the immune process.Experimental result indicated that the immune effect of microsphere loaded mucosal adjuvant was better than that of the microsphere without adjuvant,and the microsphere could stimulate the local mucosal immunity.In conclusion,the oral microsphere vaccine against CSF induced a good immune response in mice.This study provides a material basis for application of genetic engineering subunit oral vaccine in the prevention and control of CSF.