Construction Of Recombinant Lactic Acid Bacteria Expressing Tα1-IFN And Study As Immune Adjuvant

Lactic acid bacteria（LAB）are generally considered to be microorganisms that can have beneficial effects on the host when ingested in a certain dose.Because prebiotic lactic acid bacteria do not contain endotoxins,and their cell wall,nucleic acids or substances secreted by life activities have the activity of immune adjuvants,food-grade lactic acid bacteria become the preferred carrier for expressing exogenous natural immunoactive substances,so genetically engineered recombinant lactic acid bacteria are used in viral antigen presentation,heterologous protein expression,vaccine delivery vector and other fields.Objective: In this study,a recombinant lactic acid bacterium expressing thymus and interferon Tα1-IFN fusion protein was constructed by using safe food-grade Lactococcus NZ3900 as the host strain,and its immune effect as an adjuvant for ND inactivated vaccines was verified,laying a foundation for the development of a new safe vaccine immune adjuvant.Methods:（1）Construction of expression r-L.lactis-Tα1-IFN: The host bacteria NZ3900 strain was selected to construct and express Tα1-IFN recombinant lactococcus lactis using p NZ8149 expression plasmid vector without antibiotic lactose resistance labeling.The correctness of the target protein expressed was verified by Western blot,and the expression amount of the protein was quantitatively detected.（2）r-L.lactis-Tα1-IFN immunomodulatory activity in vitro: using r-L.lactis-Tα1-IFN interacts with macrophage reporter cell line J774-Dual^TM cells and PBMCs to detect their immunomodulatory activity.（3）A preliminary study of r-L.lactis-Tα1-IFN as an immune enhancer: chickens with SPF were rando MLy divided into 2 groups,and the chickens in the experimental group were given oral administration of 500 μL of r-L at 3 weeks of age and 4 weeks of age.lactis-Tα1-IFN;Chickens in the blank control group were orally administered 500 μL of PBS at 3 and 4 weeks of age,respectively.After the first vaccination 2,4 and7 weeks,anticoagulant was collected by three different stimuli of canavalin A（Con A）,ionomycin（Ionomycin）,canavalin A（Con A）,phytohemagglutinin（PMA）and lipopolysaccharide（LPS）to stimulate chicken peripheral blood mononuclear cells,detect proliferative activity,and after 7 weeks,all SPF chickens were dissected,their cecal contents were taken,and the structural changes of SPF chicken cecal flora were detected and analyzed by 16 Sr RNA high-throughput sequencing method.（4）Research on r-L.lactis-Tα1-IFN as an immune adjuvant: utilization of ultrasonic lysates of r-L.lactis-Tα1-IFN as immune adjuvants for inactivated Newcastle disease.14-day-old SPF chickens were rando MLy divided into 3groups: blank control group,vaccine control group,and recombinant r-L.lactis-Tα1-IFN oil emulsion inactivated vaccine test group,10 in each group.In the recombinant in the r-L.lactis-Tα1-IFN immunoadjuvant test group,the injection dose was 0.6 ML/p.（0.3 ML containing antigen）;In the control group of inactivated vaccine,the injection dose was 0.3 ML/p.In the blank control group,the injection dose was 0.3 ML PBS.After immunization,blood was collected 3 days,7 days,and 14 days to prepare serum;On day 14 post-immunization,collect anticoagulant blood and prepare chicken PBMCs.Results:（1）Successful construction of r-L.lactis-Tα1-IFN.The Western blot results showed that the recombinant lactococcus lactis successfully expressed the protein of interest at 25 k D,which was consistent with the expected results,and the quantitative protein detection results indicated that the expression was 27μg/ML.（2）The interaction between recombinant lactic acid bacteria expressing Tα1-IFN and J774-Dual^TMcells and PBMCs proved that r-L.lactis-Tα1-IFN can effectively activate the NF-κB and IRF signaling pathways of macrophage reporter cell lines.At the same time,it can induce chicken PBMCs to produce higher levels of IFN-γ and IL-10 and higher levels of CD80 and CD86 molecules.In vitro studies have shown that r-L.lactis-Tα1-IFN has good immunobooster activity.（3）Animal experimental results as an immune enhancer showed that recombinant r-L.lactis-Tα1-IFN immune enhancer can significantly activate antigen-presenting cells of immune chicken PBMCs and improve immune response.In the analysis of intestinal microbiota structure,r-L.lactis-Tα1-IFN increases the relative abundance of beneficial microbiota and alters the composition of the gut microbiota,specifically the probiotic genus Lactobacillus significantly increases.（4）Animal experimental results as immune adjuvants showed that in the recombinant in the trial of r-L.lactis-Tα1-IFN inactivated vaccine immune adjuvant,the expression levels of IL-2,IL-4 and IL-1β in the serum of chickens in the experimental group were effectively increased,but the expression level of IFN-γ in serum was significantly reduced.The proliferative activity of PBMCs was significantly enhanced.Antibody levels are extremely markedly increased.This showed that the recombinant Tα1-IFN immune adjuvant had good immune adjuvant activity in the trial of Newcastle disease inactivated vaccine.Conclusion: In this study,the recombinant Tα1-IFN fusion protein expressed by lactic acid bacteria showed strong immune-enhancing activity in vitro and in vivo,indicating that it has the potential to be used as an immune enhancer or immune adjuvant,which provides a basis for the research and development of new green and safe food-grade animal immune enhancers or immune adjuvants.