| Mycobacterium avium subsp. Paratuberculosis is a pathogen which can cause Johne’s disease and mainly infect ruminants, causing chronic and incurable granulomatous enteritis. The infections range of MAP is widely, including cattle, sheep and wild ruminants, for example deer. The capacity of milk production and reproductive of the animals infected with MAP all declined. It has a huge impact on arming and dairy industry and causes great economic losses.So the MAP prevention has become a major problem on our aquaculture industry. Therefore there is an urgent need to develop an efficient, economical, stable vaccine for MAP control. The surface display vaccine presents antigen on the bacterial surface, which has a unique advantage in design. Because the polypeptide antigen on bacterial surface is distinguished by immune system easily, while the outer membrane proteins, lipopolysaccharide and toxins have strong immunogenicity can be adjuvants for exogenous protein.Therefore, it has broad application prospects in the development of new vaccines.In this study, ice nucleation protein is used as the display platform to build surface display system in E. Coli.Design primer, connect the target gene to INP display platform, use PCR amplification and digest with HindⅢand NdeΙ, then connect to pET-INP vector. the protein was verified to successfully expressing and displaying on outer membrane throw western-blot, subcellular fractionation, proteinase K treatment, spots western and immunofluorescence after inducing the recombinant plasmid expressed in BL21(DE3).Recombinant strains is used to immunize Balb/C female mice, and the level of immunization is assessed by detecting cytokines, flow cytometry, serum antibody. Then attacking drug, the protective effect is assessed throw by detecting the change of cytokines, body weight and pathological changes in colon, liver and spleen. Results show that compared with the control group, the antibody level of the recombinant bacteria immunized group have a bigger rise and after the third immunization, its antibody level is 4.2 times that of vector control group. Show that the surface display vaccine carrier protein can stimulate the body to produce high levels of specific antibodies. The CD4+ and CD8+ T cells was significantly increased in display vaccine group and IFN-γ, IL-4, IL-23 / IL-17 and other cytokines were significantly increased. Show that the recombinant bacteria INP-MAP3061 c can mediate the body to produce strong cellular immune response. After infecting with MAP, we found that before 16 weeks, the rate of weight gained is increasing, and at 16-20 weeks, the rate of weight gained is decline slowly, but the rate of weight gained still keep positive, indicating that mice is keeping in growth, no weight loss. Compared the pathological results of colon, liver and spleen with the control group, we found that surface display vaccine could reduce the degree of pathological damage and slow down the course of disease.In conclusion, we use the target protein MAP3061 c with strong immunogenicity, with the help of the surface display platform on bacteria, show the target protein on the surface of E. coli. The surface display live vaccine carrier can arouse strong humoral and cellular immunity in mice and have good resistance effects for MAP infection. Which provide a new train of thought to prevention and control MAP infection and lay the foundation for the development of MAP vaccine. |
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