Study On Immune Induction And Mechanism On B Cells Of Three Bursal Peptides

Bone marrow is an important hemopoietic organ,which is important to lymphocyte differentiation and development.Thymus is considered the cellular immune center shared with both birds and mammals.In mammals,bone marrow is known as the central humoral immune organ.In mammals,bone marrow is the center of the humoral immune organ,but only as a thylakoid organ.Bursa of Fabricius(BF)is recognized humoral central immune organ unique bird,which play an important role on development and maturation of B cells.It has been reported that there are many active molecules present in BF,whereas the function and mechanism of these bursal-derived peptides on immune induction and B cell is unclear.It is of great significance for the current clinical disease prevention to further investigate the vaccine protective efficacy and mechanism of bursal-derived active molecules.In this study,two new peptides were isolated and purified from BF,and the roles on vaccine immunization with three bursal peptides were detected in mice.Furthermore,the mechanism of BHP,BP9 and BP7 on immature B cell were studied with gene microarray and Q-PCR technology.The main contents of this study include as following.1.Separation and identification of two new bursal peptidesAfter frozen,ultrasound,centrifugation and molecular sieve,the bursal extract were performed with reversed-phase high-performance liquid chromatography.The collected elution samples were analyzed with mass spectrometry.Two new peptides were purified and identified with amino acid sequences of LMTFRNEGT and GGCDGAA,and were named as BP9 and BP7,respectively,which enriched the bursal peptide library and laid a foundation for further study.With the homologous protein analysis,BP9 was similar to gamma-interferon-inducible lysosomal thiol reductase and suppressor of cytokine signaling 7.BP7 was similar to interferon-induced helicase C domain-containing protein 1 and immunoglobulin heavy chain variable region.These data suggested that these two newly peptides might be involved in immune response and other biological functions.2.The immune regulation of bursal peptides on vaccine and immunizationTo investigate the immunomodulatory activity on immune induction,mice were immunized with vaccine and BHP,BP9 or BP7,to detect antibody level,spleen lymphocyte proliferation and T cell subtype,respectively.First,mice were immunized with JEV vaccine and BHP from O.Olmg/mL to 0.25mg/mL to detect antibody response and cellular immune response,respectively.The results showed that BHP coimmunization with JEV vaccine generated significant increased antibody levels,neutralizing antibody titers and spleen lymphocyte viability,compared to that of vaccine control.Also,the subpopulations of T cells in spleen lymphocytes were significantly modified in the mice immunized with JEV vaccine and BHP.Second,mice immunized with JEV vaccine and BP9 produced significantly increased antibody levels,including special to JEV,antibody subtypes IgGl and IgG2,and neutralizing antibody.Unexpected,antibody levels were decreased following the increased BP9 concentrations,in which antibody level produced from mice immunized with 0.01 mg/mLBP9 were highest among all experimental groups.Although the increased lymphocyte viability,T cell subtype were not significantly different after BP9 immunization.Third,mice immunization confirmed that mice immunized with AIV antigen and BP7 produced the decreased antibody levels with the increased concentrations of BP7,whereas antibody levels of serum from mice immunized with AIV vaccine and BP7 at experimental concentrations were not significantly different.Furthermore,BP7 played the different immune induction roles on IgG2a and Thl/Th2 type cytokine productions in mice immunized with AIV antigen and vaccine.The experimental results showed that these three bursal peptides had different immunomodulatory activity on vaccine immunization,which were related to concentrations and immunogenic type.3.The mechanism of bursal-derived peptides on mouse pre-B cellsIn order to study the effect of bursal-derived peptides on mice immature B cells,in this paper,mouse immature B cell WEHI231 cell was used as a model.The results of MTT assay showed that BHP and BP7 could inhibit WEHI231 cell viabilities,whereas BP7 treatment played no significant effect on WEHI231 cell viabilities.Furthermore,gene microarray was used to detect the gene profiles of WEHI231 cells after 0.01μg/mL BHP,0.01μg/mL BP9 and 0.01μg/mL BP7 treatment,respectively.Gene chip results showed that there were 1539,2602 and 2465 gene differential expressions in WEHI231 cells treated with BHP,BP9 and BP7,respectively.Functional analysis and pathway analysis revealed that these regulated genes were involved in many pathways and biological processes,including immune related processes,autophagy,migration,and metabolism.Pathway network analysis showed that the regulated genes with BHP treatment were involved various physiological metabolic pathways and signaling pathways,including TCA cycle,Glycolysis,p53 signaling pathway and mTOR signaling pathway.Endocytosis and ubiquitin mediated proteolysis might be close to other involved pathways after BP9 treatment in immature B cell.Furthermore,ubiquitin mediated proteolysis and antigen processing and presentation might be the important pathways after BP7 treatment.Functional analyses showed that the differential genes with BHP treatment were involved various immune related biological functions,including proliferation and activation of lymphocyte and T cell,T cell mediated immunity and regulation of adaptive immune response.Also,the regulated gene with BP9 treatment were involved the mainly related immune responses,including T cell activation,cytokine secretion,MHC molecular,and ubiquitin related biological processes.The differentially-expressed genes with BP7 treatment were involved in T cell and lymphocyte proliferation,Th2 cell differentiation,antigen processing and presentation,MHC class II and I biosynthetic process,B cell activation and mediated immunity,and cytokine.These results showed the potential mechanism of bursal-derived peptides on immature B cells development.Briefly,the active peptides from bursa have important effect on vaccine immunization and various biological processes.Further study on the mechanism of bursal-derived peptides on immune induction and B cell development is helpful to explain the process of B cell differentiation,and also helpful to understand the physiological metabolic processes and associated genetic diseases,which has guiding significance for clinical application.