| Tuberculosis(TB), caused by Mycobacterium tuberculosis(MTB), is a livestock and poultry comorbid chronic wasting diseases, with HIV / AIDS and malaria consisting of the three major threat to the health of people all over the world. In 2013, 9 million people were suffering from tuberculosis, and 1 million 500 thousand people died of the disease. With the spread of drug resistant Mycobacterium tuberculosis and HIV / AIDS, the control and prevention of tuberculosis is facing great challenges. In early study, we found that there are some limitations of several vaccines against tuberculosis. Vaccination with Bacille Calmette Guerin(BCG) is the main way of the prevention of tuberculosis, but there are low efficiency and limitation of protection; the effectiveness of DNA vaccine remains to be improved; and the immune response of subunit vaccine is of short duration, so its application is limited. Therefore, the development of efficient, safe and widely used model combined with the vaccine is imminent. Therefore, we constructed a bacterial surface display vector to display the PepA, PPE18 and EsxH protein of Mycobacterium tuberculosis on the surface of Escherichia coli, then study the immune effect and evaluate the potential of the constructed live vector vaccine in the prevention and control of TB.In this study, we cloned three exogenous protein(PepA, PPE18 and EsxH) encoding gene(rv0125, rv1196, rv0288) and three fusion gene(rv0288-1196-0125) to the INP based on the successful surface display of the EGFP surface display, transformed to E. coli and then verified the expression and position of foreign proteins through different means. Immunoblotting results showed that the three encoding genes and the fusion gene were detectable of protein bands; subcellular fractions result showed that the proteins were located in the cell membrane and cell wall; after the proteinase K digestion, the protein cleavage into many small protein fragments; and the specific fluorescence signal of the bacterial surface could be observed under the laser scanning confocal microscope. At last, we take mice as animal model injected with the induced recombinant bacteria by intraperitoneal, taking serum, isolating spleen lymphocytes regularly and then detecting of antibody titer, the level of cytokine and CD4+or CD8+, and analyzing the immunity. The results are as follows: compared to control group, the serum antibody levels of immune groups were greatly improved. Two weeks after the third immunization, the antibody level of INP-0288 group can reach 4 times compared to the empty plasmid control group, even if the lowest antibody level of INP-0288-1196-0125 increased 2 times compared to the empty plasmid control group. It showed that the live vaccine vector of surface display could stimulate the body to produce a high level of specific antibody against the displayed protein. The CD4+ T cells number of all immunized groups showed a rising trend; and for CD8 + T cells, INP-0288 showed rising trend, the other three groups a slight decline after increase, both higher than the control group. INP-0288 group of CD4+ / CD8+ showed increased after decreased trend. The other three groups show a decline after rising trend. The cytokine levels exists significant difference: for IFNγ, the level of the immune group showed a decreasing trend, lower than the blank group; for IL-4, the level of each immune group was significantly increased at different time points; for IL-17 A, compared to the blank group, the INP-0288 group was significantly higher, while the other three groups showed a lower level.In summary, using the constructed surface display platform INP we displayed the PepA, PPE18, and EsxH and the fusion protein of Mycobacterium tuberculosis on the surface of E. coli, and the constructed three kinds of protein surface display live vector vaccine were able to stimulate strong humoral and cellular immunity in mice. This study lays a good foundation for the development of new Tuberculosis vaccine, and provids a new way of thinking for the prevention and control of Tuberculosis. |
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