Study On Recombinant Lactic Acid Bacteria Expressing Avian β-defensin As Immune Adjuvant

Probiotic lactic acid bacteria(LAB)are large,without endotoxin,can adsorb on mucosal cells and settle on the mucosal surface.Therefore,lactic acid bacteria are the preferred carrier for expressing and delivering therapeutic and prophylactic foreign proteins.Avian beta-defensins(AvBDs)are one of the natural antibacterial peptides mainly present in the natural mucosal system,which mainly exerts anti-infection,broad-spectrum antibacterial and wide-ranging immunomodulatory effects,and avian β-defensin is a small cationic non-glycosylated polypeptide,which is very suitable for prokaryotic expression.Therefore,this study used the food-grade Lactococcus lactis NZ3900 host strain to construct a complete food-grade recombinant lactic acid bacterium expressing avian β-defensin.In this study,three sets of recombinant lactic acid bacteria expressing recombinant avian β-defensin were constructed using three expression plasmids and food-grade host strains.The expression of avian β-defensin 1(AvBD1),avian β-defensin 2(AvBD2),avian β-defensin 6(AvBD6),and avian β-defensin 13(AvBD13)and fused avian beta-defensin 1/2/6/13 functional fragment composed of fused avian beta-defensin AvBD12613 were constructed,a total of 11 strains of recombinant lactic acid bacteria.Among them,the expression level of 5 recombinant lactic acid bacteria is stable and the recombinant protein size is correct.It is the recombinant lactic acid bacteria used in this study for in vitro screening and in vivo animal test verification.Using the interaction of the bacterial lysate mixture of recombinant lactic acid bacteria expressing avian β-defensin with macrophages,peripheral blood mononuclear cells to detect the immunomodulatory activity of the constructed recombinant lactic acid bacteria in vitro.In vitro screening test results showed that recombinant avian β-defensin(recombinant lactic acid bacteria r-L.Lactis-AvBD12613 cell lysis mixture)can significantly activate the IRF and NF-κB signaling pathways of the macrophage reporter cell line J774-Dual ? cells;Five recombinant avian beta-defensins rAvBD1,rAvBD2,rAvBD6,rAvBD13 and rAvBD12613 can induce HD11 in chicken macrophage cell line to produce higher levels of IL-10;of which rAvBD12613 can also induce HD11 to express higher levels of CD80 and CD86 molecule.Recombinant avian beta-defensins rAvBD12613,rAvBD1 and rAvBD6 can induce chicken peripheral blood mononuclear cells(PBMCs)to produce higher levels of IL-12 and IL-10;Among them,rAvBD12613 can induce chicken PBMCs to express higher levels of CD80 and CD86 molecules.In vitro screening shows that recombinant avian defensin r-L.Lactis-AvBD12613 has strong immunomodulatory activity.Using the cell lysis mixture of recombinant lactic acid bacteria expressing the combined avian defensin rAvBD12613 as an immune adjuvant for Newcastle disease live vaccine and Newcastle disease inactivated vaccine.The immune adjuvant activity of the combined avian defensin rAvBD12613 was verified by measuring the expression levels of cytokines in serum,the proliferation activity of PBMCs and peripheral blood leukocytes,and antibody levels.The animal experiment results show that: in the recombinant avian beta-defensin rAvBD12613 live adjuvant vaccine test,one week after injection,the expression levels of IL-4 and IL-1β in chicken serum of the experimental group were significantly increased,while the serum levels of IL-10 and IFN-γ are not significantly different,compared with the wild bacteria control group and the live vaccine control group;after three weeks of immunization and one week after challenge,the antibody level of the experimental group was significantly increased;After three weeks of immunization and one week of challenge,the proliferation activity of chicken peripheral blood mononuclear cells(PBMCs)in the experimental group was significantly improved;After three weeks of immunization and one week of challenge,the proliferation or cell activity of peripheral blood mononuclear cells(PBMCs)of chickens in the experimental group was significantly improved.In the inactivated vaccine test,after three weeks of immunization and one week after challenge,the antibody level of the experimental group can be significantly increased;After three weeks of immunization and one week of challenge,the proliferative activity of chicken peripheral blood mononuclear cells(PBMCs)and white blood cells in the experimental group can be significantly improved,which is significantly higher than that of the inactivated vaccine control group.The results showed that the recombinant avian beta-defensin rAvBD12613 immune adjuvant had good immune adjuvant activity in both Newcastle disease live vaccine and inactivated vaccine.The recombinant avian β-defensin rAvBD12613(r-L.Lactis-AvBD12613)constructed in this study showed strong immunomodulatory activity in vivo and in vitro experiment,and has the potential to be an immune adjuvant or immune enhancer.It laid the foundation for the research and development of new food safety animal immune adjuvants or immune enhancers.