Optimization Of Saponin Immune Adjuvant And Its Delivery System And Immune Effects Research

Saponins are amphoteric plant glycosides.They have been widely used in food,agriculture,medical treatment and so on.Quil A is a kind of triterpene saponin isolated from saponin tree in South America.As the most effective saponin immune adjuvant component,it has been widely studied and confirmed.It is the immune adjuvant that can not only stimulate Th1 type cellular immune response,but also induce the production cytotoxic T lymphocytes(CTL)in the body.However,due to its heterogeneity,instability,hemolytic activity certain toxic and side effects,it is necessary to form an adjuvant system with the delivery agent or find other alternative components.In this study,a variety of saponins were tested as possible adjuvants,and hydroxypropyl-β-cyclodextrin(HP-β-CD)or lecithin with cholesterol delivery system were used as delivery agents to optimize with saponins and to study their immune effects.In this study,ovbumin(OVA)was used as antigen,and different saponins and delivery agents were combined as optimized immune adjuvants.The antibody levels after immunization were tested by ELISA.The results showed that the immune system formed with HP-β-CD and Quil A stimulated high levels of antibodies,which the OVA specific IgG,IgG1 and IgG2a are about 2,2 and 1.3 times higher than those immunized with Quil A alone,and is significantly different from those immunized with OVA alone(P<0.01).These demonstrate that HP-β-CD maintained and enhanced the adjuvant activity and properties of Quil A.In addition,both Tet and Cel showed adjuvant activity,among which,Cel and Del delivery agent had better immune effect.Both these saponins can significantly improve antigen-specific IgG and IgG1,moreover Cel alone can significantly improve antigen-specific IgG2aThe proliferation of lymphocytes was detected by MTT assay.Tet、Cel、HP-β-CD and the delivery system composed of lecithin and cholesterol can promote the proliferation of lymphocytes after OVA immunized mice induced by Con A,indicating that the delivery system can significantly enhance the cellular immune response of immunized mice.Flow cytometry was used to detect the changes of splenic lymphocytes in the immunized mice.The results showed that Both Tet and Cel could increase the number of CD19~+lymphocytes.However,The number of CD4~+lymphocytes decreased in the delivery system consisting of adjuvant and delivery agent,which was significantly different from that in the OVA group(P<0.01),while the number of CD19~+lymphocytes remained unchanged,suggesting that both HP-β-CD and lecithin cholesterol may promote CD4~+lymphocytes migration to peripheral lymphoid tissue,thereby mediating enhanced antibody response.Real-time PCR results showed that the expression of BCL6 gene was up-regulated when saponins with adjuvant activity.The expression of BCL6 gene was down-regulated when saponins with adjuvant activity were combined with delivery agent,suggesting that the expression of BCL6 gene was correlated with saponins alone or with delivery agent.The above experimental results laid a foundation for selecting suitable saponins as effective immune adjuvants,and laid a certain research foundation for the effects of HP-β-CD as saponins adjuvant delivery agent,also provided a new idea for the wide application of saponins immune adjuvants.