| CD40(cluster of differention 40)is a member of the tumor necrosis factor receptor superfamily.It belongs to the type I transmembrane glycoprotein and is mainly expressed on the surface of epithelial cells,endothelial cells,fibroblasts,hematopoietic cells,some tumor cells and immune cells.CD40 on the surface of immune cells binds to CD40 L produced by activated T cells,which can promote the proliferation,differentiation and maturation of DCs,enhance their antigen presenting ability and ability to phagocytize apoptotic cells;CD40-CD40 L can be used as the second signal for B cell activation,which participates in the process of B cell activation,proliferation,and differentiation,promotes the production of antibody and type conversion,promotes the production of memory B cells,and enables the B cells to be apoptotic to survive and proliferate,forming a germinal center;it can obviously up-regulate the expression of CD80 and CD86 molecules in APC cells.As the second signal necessary for T cell activation,it can effectively stimulate the activation of the body's immune system.For a long time,the lack of effective early pregnancy detection methods has been a major problem restricting the development of the dairy industry,which can lead to problems such as long empty periods,low reproduction rates,and increased costs.Cow early pregnancy factor(EPF)is one of the earliest recognized biochemical markers of pregnancy,so the presence or absence of EPF can be used as a reliable indicator of early pregnancy.According to the EPF epitope sequence,a B-cell epitope peptide composed of 14 amino acids was designed and synthesized in the early stage of this project.The B-cell epitope peptide belongs to the small molecule hapten.Due to the weak immunogenicity of small molecules,the immune process needs to be coupled to large carrier proteins,and the commonly used Freund's adjuvants are toxic and difficult to be absorbed.Therefore,the preparation of small molecule antibody is difficult.An immunoadjuvant for small molecules that requires no coupling,no emulsification,and is easy to use.Many studies have shown that CD40 antibody can mimic the binding of CD40 L to CD40 on the surface of B cells,play a biological function similar to CD40-CD40 L binding,and promote B cell activation to produce antibody.Based on the above background,this experiment used a designed and synthesized EPF B-cell epitope peptide as an immunogen to preliminary explore the immune-enhancing effect of CD40 antibody on small molecule peptides,and lays a foundation for the development of a new type of adjuvant against hapten immunity.First,bioinformatics analysis of the extracellular segment of CD40 in BALB / c mice with DNA Star was performed to predict its antigenicity;then prokaryotic expression and purification of the extracellular segment of CD40 in BALB / c mice;CD40 antibody was purified and analyzed for applicability.Finally,BALB / c mice were co-immunized with the CD40 antibody and the B-cell epitope peptide.By measuring the changes of serum antibody levels in different periods of each group of mice and the spleen B cell CD molecules and related cytokines at the mRNA or protein level Differential expression to analyze the effect of CD40 antibody on the immune effect of B-cell epitope peptidesThe results showed that the secondary structure of the extracellular segment of CD40 was analyzed by DNA Star,and the epitope was predicted to be at amino acid residues 5-29,32-29,50-101,114-130,136-163,and 168-173,strong antigenicity,can be used as a good immunogen for antibody production.The recombinant protein of the extracellular domain of CD40 was successfully expressed and purified.The rabbit was immunized,and the CD40 polyclonal antibody was successfully prepared and purified.It can specifically bind to mouse CD40 natural protein and has good applicability.Immunoenhancement experiments showed that 10 ?g and 20 ?g CD40 antibody increased the B cell epitope peptide antibody level significantly,and 2 ?g CD40 antibody was less effective in enhancing immunity.The results of real-time quantitative PCR showed that at the mRNA level: the expressions of CD19,CD86,CD138,IL-4 and IL-5 were significantly up-regulated in the 10 ?g and 20 ?g CD40 antibody groups at each stage;IL-2 and IFN-? were elevated but not significant in each group;which indicates that 10 ?g and 20 ?g CD40 antibody caused significant B cell proliferation and differentiate into a large number of plasma cells,and mainly humoral immune response.ELISA testing results showed that at the level of serum,the expression of IL-4 and IL-5 was significantly up-regulated in the 10 ?g and 20 ?g CD40 antibody groups at each stage,and IFN-? also showed a small increase in expression;IL-2 and IFN-? were elevated but not significant in each group.The results showed that 10?g and 20?g CD40 antibody caused significant Th2 type immune response,and also caused the slight Th1 type immune response,but the main is Th2 type immune response.In summary,the CD40 antibody prepared in this experiment at a dose of 10 ?g or 20 ?g / piece / time caused a significant serum antibody promotion effect on the designed and synthesized B cell epitope peptide,and the immune response occurring in the body was mainly the Th2 type humoral immune response,which provides for the experimental basis for further exploring the immune mechanism of CD40 antibodies to auxiliary small molecule substances and the development of new adjuvants. |
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