Molecular Basis Of BP7 Regulating Chicken Immature B Cells And Immune Function Of Chicken MDA5

The bursa of fabricius(Bursa of fabricius,BF)is located near the cloaca of poultry,which is a unique central immune organ of poultry,and also is the place where B lymphocytes develop.Bursal-derived peptides are a series of active molecules derived from BF that regulate the immune response.There were researches on the biological functions of the Bursal-derived peptides BP1,BP3 and BP5,such as anti-tumor effects,regulation of avian immune response and enhancement of the effectiveness of inactivated avian influenza vaccines.BP7 is recent reported active hepeptide that can promote antibody response and B cell autophagy.There were few reports about BP7 on the immune regulation of avian immature B cells.Chicken MDA5 is a BP7 homologous protein,a member of the RLRs(including RIG-I and MDA5)family,which can recognize viral RNA invading cells and activate downstream interferon regulatory pathways to inhibit viral replication.In waterfowl,RIG-I recognizes the infected AIV virus.However,the chicken lack of RIG-I,and MDA5 play an important role in the recognition of AIV infection.This article preliminarily showed that BP7 could promote the maturation of immature B cells and participated in a variety of immune-related pathways.BP7 homologous protein MDA5 fragment MDA5-2 had strong antiviral effect,and recombinant proteins MDA5-1 and MDA5-2 could be used as an immune enhancer to improve the immune efficiency of the H9N2 inactivated vaccine.The main research contents were as followed:1.The gene expression profiles and function analysis of chicken immature B cells treated with BP7The aim of this study was to explore the molecular basis of the regulation of immature chicken B cells by bursal-derived peptide BP7.The m RNA level of Ig M was detected by RTq PCR,and the gene expression profiles and biological functions were analyzed by c DNA microarray.The results showed that the m RNA level of Ig M in DT40 cells stimulated by BP7 was significantly increased.Microarray analysis showed that there were 1345 differentially expressed genes in DT40 cells treated with BP7,which were involved in 17 pathways,including receptor interaction,signaling pathways,metabolic and proteolysis related pathways.The network analysis of pathway showed that cytokine-cytokine receptor interaction was the key pathway in DT40 cells stimulated by BP7.Go function analysis showed that the immune related biological functions in DT40 cells stimulated by BP7 mainly included immune response,immune response signal,Th1 type immune response,production and regulation of cytokines,and cytokines’ receptor activity.This study elucidated the molecular basis of BP7 regulating avian immature B cells.These results also provided new insights for the mechanism of BP7 on B cell differentiation regulation,and provided an important basis for the development of new vaccination adjuvant.2.Effect of MDA5 fragment on innate immunityIn this study,a fluorescent quantitative PCR method for the NA gene of the H9N2 virus was established.Two fragments of the BP7 homologous protein MDA5 gene were designed through Protean software,and named MDA5-1 and MDA5-2 respectively,which were constructed into two eukaryotic expression plasmids Flag-MDA5-1 and Flag-MDA5-2.After the recombinant plasmids transfected into the cells,the cells were infected with H9N2 virus,and the inhibitory effects of the two fragments of MDA5-1 and MDA5-2 on H9N2 virus replication were detected.RT-q PCR experiments showed that MDA5-2 could increase the expression of antiviral molecules IFN-β and PKR,and activated the downstream molecule MAVS,and also inhibited the expression of viral proteins HA,NA and NS.Using RNA interference technology to knock down the MDA5 and MAVS genes,and then transfected the plasmids MDA5-1 and MDA5-2,we found that after knocking down MDA5 and MAVS,the virus replication was enhanced.After transfecting the MDA5-1 plasmid,the expression of IFN-β did not increase significantly and virus replication was enhanced.After transfecting the MDA5-2 plasmid,the expression of IFN-β was significantly increased and viral replication was inhibited.This showed that MDA5-2 had a stronger antiviral effect on H9N2 AIV than MDA5-1.This study illustrated that the MDA5-2 gene might participate in the antiviral pathway,and could activate the expression of IFN-β and inhibit viral replication,which provided the important basis for further studying the immune function of the MDA5 protein.3.Effect of MDA5 fragment on H9N2 inactivated virus vaccineIt was reported that PPRs was used as a new type of immune adjuvant to improve vaccine immunity.MDA5 is belonged to one of PPRs,and the CARD region is the key region for interaction with downstream molecules.In order to explore whether the recombinant proteins of MDA5-1 and MDA5-2 could be used as immune enhancer to improve the immune efficiency of H9N2 inactivated virus vaccines,prokaryotic expression plasmids His-MDA5-1 and His-MDA5-2 were constructed and expressed in this experiment.The purified proteins MDA5-1 and MDA5-2 were mixed with oil adjuvant and H9N2 inactivated virus vaccine to immunize SPF chickens.The sera of the immunized chicken were collected on the 7th day after each immunization to detect antibody titer.The results showed that compared with the BSA control group,the HI antibody titer of chicken immunized with two recombinant protein groups were increased after immunization,and the serum Ig M and Ig Y levels were also slightly increased.The m RNA expression levels of the surface marker molecules of T lymphocytes and B lymphocytes after immunization with recombinant protein His-MDA5-2combined with white oil adjuvant and H9N2 inactivated virus vaccine were higher than those with recombinant protein His-MDA5-1,but there was no significant difference.The study showed that the recombinant proteins of MDA5-1 and MDA5-2 could enhance the immune efficiency of H9N2 inactivated virus vaccines,but the amount of protein needed to be further explored.This experiment provided the important experimental materials for further research on the immunoregulatory function of MDA5 protein and further clinical application.