Construction Of The Surface Display Carrier Vaccine Of Mycobacterium Avium Subsp.paratuberculosis Antigen Protein And Evaluation Of Its Immune Efficacy

Paratuberculosis is a chronic wasting infectious disease and characterized by intermittent diarrhea and granulomatous enteritis.It is caused by Mycobacterium avium subsp.Paratuberculosis(MAP).The disease is widespread worldwide,and the affected animals usually show clinical symptoms such as gradual weight loss and decreased production performance,which brings huge economic losses to the animal husbandry industry.Due to the lack of an effective treatment plan and the current vaccines have significant limitation,it is urgent to develop a novel vaccine with good immune effect.In this study,ice nucleation protein(INP)was used as the carrier protein,and the MAP antigen protein was carried and positioned on the surface of E.coli by using the function displayed of INP,and then the immune protective efficacy is studied by mice model.In this study,the four MAP genes map1761c,map1636c,map1589c and map3840 were inserted into p ET-INP vector,respectively,and the resulting plasmids were transferred into E.coli BL21(DE3),Western blot,separation of bacterial outer membrane components,bacterial immunofluorescence and immunoelectron microscopy techniques were used to verify the expression and localization of the target protein.The C57BL/6 female mice were divided into six groups.The PBS group,INP group and INP3840group were used as the control group.After 2 weeks of first immunization,a booster immunization was performed.Serum was collected regularly to detect specific Ig G antibody and cytokine levels,and the spleen lymphocytes were detected the number of CD4~+T and CD8~+T cells,the level of cytokine transcription,and the level of lymphocyte proliferation.Four weeks after the second immunization,the mice were infected with the MAP K-10 strain,and the spleen,liver and colon pathological damage and the bacterial load were comprehensively evaluated for the protective efficacy.Western blot results showed that the target protein is expressed and the target band appears at the expected size;the separation of bacterial outer membrane components showed that the target protein can be detected on the bacterial outer membrane;the bacterial immunofluorescence result showed that under an inverted fluorescence microscope immunostained bacteria can present specific fluorescent signals;the results of immunoelectron microscopy show that specific colloidal gold particle labels can be observed in the outer membrane of the bacteria.The serum antibody results showed that INP1589c and INP3840groups can induce high levels of specific Ig G antibodies;the lymphocyte proliferation results showed that the lymphocyte proliferation level of the INP1589c and INP3840 groups were significantly increased 4weeks after immunization,while the INP1761c and INP1636c groups had no significant difference compared with the PBS and INP groups;T cell subpopulation results showed that the spleen CD4~+T lymphocytes and the ratio of CD4~+T/CD8~+T cells were significantly increased in the INP1589c group and INP3840 group mice since the 4 weeks after immunization,while the INP1761c and INP1636c groups had no significant difference compared with the PBS and INP groups;the cytokine results showed that the INP1589c and INP3840 groups mainly induced Th1 Type cytokine(IFN-γ,IL-2)-based immune response.The results of tissue load and pathological damage showed that the mice in the INP1589c group and the INP3840 group were better able to resist MAP infection,while the INP1761c group and the INP1636c group had relatively weak protective effects.In this study,INP was used as a display platform to construct a vaccine carrier.The INP1589c immune group can effectively stimulate mice to produce better humoral and cellular immunity,and has a good resistance to MAP.It proves the feasibility of the surface display carrier vaccine,and a new idea for the development of a novel paratuberculosis vaccine.