Functional Mechanism Of Bursal Active Peptides Regulating B Cell Differentiation And Maturation

The prevention and control of animal diseases plays an extremely important role in the healthy development of animal husbandry.Vaccine immunization is not only the main measure for the prevention and control of infectious diseases of livestock and poultry,but also an primary strategy of animal disease prevention and control plan.Most vaccines play a good clinically role with adjuvants.Therefore,the research and technological innovation of safe,residue-free,and effective new immune enhancers have become an urgent problem in the development of aquaculture industry.The bursa of Fabricius(BF)is currently recognized as a central humoral immune organ and a key site for the differentiation and maturation of B cells.B cells are also key cells that mediate the humoral immune response.Studies have shown that the bursa-derived active peptide could promote the immune response and humoral immune response.However,the mechanism of the bursa-derived active peptides in regulating B cells differentiation is still unclear.Therefore,the functional mechanism research of bursa-derived active peptide on the differentiation of B cells will have the important guiding significance for studying the active molecules in the humoral immune system and promoting the immune protection effect of vaccines for the current clinical disease prevention and treatment.In this thesis,three bursal active peptides of BP5,BP7 and BP9 are immunized with avian influenza vaccine,and three bursal active peptides are used to stimulate the spleen cells of avian influenza-immunized mice in vitro to explore the regulation of the bursal active peptides in vivo and in vitro.The functional mechanism of B cell differentiation and maturation proved through the detection of serum antibody levels,lymphocyte activity and other experiments,respectively.Then,the functions of the active peptide of the bursa of Fabricius on the cells of each stage of the mouse B cell development were studied.Also,in order to understand the molecular mechanism of the bursa active peptides regulating the development of B cells,B cells were isolated and purified,and RNA-Seq high-throughput sequencing technology was used to determine the m RNA transcriptome changes in the purified B cells.These results would provide a new idea for further study on the role of bursal active peptides in the immune system,and lay a good foundation for the application of active peptides from the experimental stage to practical application.The main researchs were as follows.1.Effect of bursal active peptide on humoral immune B cells in vitroIn order to further explore the functional mechanism of bursal active peptides on B cells,mice were immunized with avian influenza vaccine.The spleen cells of immunized mice were stimulated in vitro with BP5,BP7,and BP9 at 0.01,0.1,and 1μg/m L respectively,and T cell subtypes,T cell apoptosis,cytokines,B cell subtypes,and phosphorylated protein expression in the stimulated B cells were detected.The experimental results showed that bursal active peptides BP7 and BP9 increased the level of activated T cells,and promoted the differentiation of T cells into CD3~+CD8~+T cell subtypes,and inhibited the early apoptosis of T cells.BP7 promoted the secretion of splenic lymphocytes IL-4 and IFN-γ.Also,three bursal active peptides could stimulate the proliferation activity of splenic lymphocytes,and BP5 and BP7 promoted the level of total B cells(CD19~+)and stimulated the proliferation of differentiated B cells and plasma cells.Also,BP5 could promote the proliferation of activated and mature B cells.Furthermore both BP7 and BP9 increased the expression of m TOR(S2448)phosphorylated protein,and BP9 could promote the expression of STAT3(Y705)phosphorylation protein.The above results indicated that bursal active peptide might not only promote the development of B cells in vitro,also participate in the regulation of T cell response and activation signal pathways.However,its mechanism of action and active peptide dosage still need to be further explored.2.Regulation of bursal-derived peptides on B cell differentiation in vivoIn order to explore the functional mechanism of bursal-derived peptides on B cells differentiation,BP5,BP7,and BP9 were selected at 0.01mg/m L,0.05mg/m L and0.25mg/m L three concentrations to be mixed with inactivated avian influenza vaccines.Six-week-old ICR mice were immunized twice by intraperitoneal injection,and PBS and vaccine control group were set up at the same time.Experimental results showed that BP5,BP7,and BP9 significantly promote the increase of antibody levels,lymphocyte proliferation activity and activated B cell proliferation in mice,however three polypeptides have different function on the regulation of B cell subtypes.BP5 could promote the levels of differentiated B cells(CD19~+CD43~+)and mature B cells(CD19~+Ig D~+),and reduced the levels of undifferentiated B cells and immature B cells in the immunized mice.BP7 and BP9 promoted total B cells(CD19~+)proliferation,and BP7 could promote the increase in the level of CD19~+CD38~+labeled B cells,and BP9 stimulated the increase in the level of plasma cells in the immunized mice.Additionally,BP5 promoted the proliferation and differentiation of CD3~+T cells.These results indicated that these three bursal active peptides could be used as the immunologically active substances to regulate the differentiation and proliferation of B cells in mammals.However,the mechanism of action between the regulation of B cell function and the immune dose still needed to be further explored.3.Bursal active peptide regulates B cell transcriptomeThe bursa of Fabricius plays an important role in the development of B lymphocytes and is controlled by a variety of m RNAs,which are expressed in different tissues of eukaryotes.In order to have a deeper understanding of the molecular mechanism by which the bursa of fabric active peptides regulate the development of B cells in mammals,based on the experimental results in Chapter 2,we selected 0.05mg/m L BP5,0.25mg/m L BP5,and 0.01mg/m L BP9 combined with avian influenza vaccine in the immunization group to detect the m RNA profiles of B cells isolated from the immunized mice with RNA sequencing technology.Differential expression analysis showed that 0.05mg/m L BP5,0.25mg/m L BP5,and 0.01mg/m L BP9 immunization groups could regulate 1128,632,and4093 differentially expressed genes in the immunized B cells,and differential m RNA genes were randomly selected for fluorescence quantitative PCR detection to verify.The results are consistent with the transcriptome analysis results,indicating that the transcriptome results are accurate and reproducible.Then the differential m RNA genes were analyzed for the significance of GO and KEGG.The results of GO and KEGG in the BP5 experimental group showed that the differential gene products were mainly distributed in cells,cell membranes,organs and other components,which mainly performed molecular functions,and biological processes,such as binding activity cellular processes and biological regulation,which play a mediating role in biological processes such as antigen presentation and response to stimuli.However,the adjustment methods of BP5 at different doses are slightly different.GO and KEGG results of BP9 experimental group showed that the differential expressed genes were mainly distributed in cells,cell membranes,organs and other components,which had catalytic activity molecular functions,and were mainly related to the metabolic processes of cytokines,proteins and amino acids.These results indicated that the bursal active peptides could regulate m RNA genes expression involved in the process of regulating the development of B cells,which provided an idea for the study of the bursal active peptides in regulating the development of B cells at the gene level.Furthermore,bursal-derived peptides,as a new type of immune enhancer,might provide an important experimental basis for improving vaccine immunity.