Construction And Immunogenicity Study Of L7/L12, Omp31 And L7/L12-omp31 Fusion Gene Eukaryotic Expression Vector Of Brucella

Brucellosis is one of the most common bacterial infectious diseases, infecting more than200 kinds of poultry, livestock, wildlife and people in global scope. Human or animal brucellosis has spread in160 countries and regions, resulting in huge economic losses. Vaccine inoculation is an effective measure to control brucellosis. Commonly used animal vaccines include live attenuated vaccineS19、S2、M5、Rev-1 with better protective effect, good stability and simple way to vaccinate. The attenuated vaccine may be a "throwback",causing some security problems, and the diseases caused by vaccines may be hardly distinguished from that by natural infections; in order to overcome the shortcomings of these vaccines, it is eager to develop new vaccines. Therefore, according to the L7/L12 and Omp31 genes of Brucella, this paper carried out the DNA vaccines, hoping to provide a new theoretical basis in vaccine development.( 1) Construction of L7/L12, Omp31 and L7/L12-Omp31 fusion genes of Brucella eukaryotic expressing vectorThe experiment amplified L7/L12 and Omp31 genes using brucella suis S2 genome as template, and then adopted overlap extension PCR method to amplify L7/L12-Omp31 fusion genes, connected with cloning vector pGEM-T respectively. After identified correctly the plasmids were connected with the enzyme digestion of pVAX1 vector, to construct eukaryotic expressing plasmids pVAX1- L7/L12, pVAX1- Omp31 and pVAX1- L7/L12- Omp31.(2)Eukaryotic expression of L7/L12, Omp31 and L7/L12- Omp31 fusion gene of BrucellaThe pVAX1- L7/L12, pVAX1- Omp31, pVAX1- L7/L12- Omp31 eukaryotic expressing vector were transfected into Vero cells by liposome mediated transfection,and detected with indirect immunofluorescence and Western blotting after 48 h. The results show that the L7/L12,Omp31 and L7/L12-Omp31 fusion genes express in Vero cells and the interest protein has an ideal reactionogenicity.( 3) Immunogenicity analysis of L7/L12, Omp31 and L7/L12-Omp31 fusion gene expression products of BrucellaWith a large extracted of plasmids L7/L12, Omp31 and L7/L12-Omp31 as DNA vaccines were used to kun-ming mices, and evaluated the humoral and cellular immunity of the immunity of the immunized mice by detecting the level of antibody, After the three immunization using indirect ELISA for the detection of antibody level in mouse serum, all the experimental groups produced specific anti-Brucella antibody; CCK8 detected with ConA and S2 stimulated the proliferation of spleen lymphocytes in mice after immunization, S2 stimulation index higherthan the ConA of the stimulus index; using the flow cytometry to detect the classification of the spleen T lymphocyte subgroup,the CD4+/ CD8+ ratio of the experimental group is less than that of the PBS group, indicating that the immune response is mainly mediated by CD8+. The enzyme linked immunosorbent assay with double antibody sandwich method was adopted to examine the secretion level of the immune mouse serum IFN-γ, IL-4, IL-10 and IL-12 cytokines,indicating that IFN-γ, IL-4, IL-10 and IL-12 cytokine levels increased in the mouse serum, but IL-4 value is much lower compared to other cytokine values. Showing that the DNA vaccine can induce the mice to produce immune responses of Th1 and Th2 type cell, but Th1 type trends to be major one.In this study, pVAX1- L7/L12, pVAX1- Omp31 and pVAX1- L7/L12- Omp31 was constructed as the nucleic acid vaccine to immune mice, comprehensive analysis of humoral and cellular immunity indicatesd that the L7/L12-Omp31 fusion gene DNA vaccine immune effect is better than single gene DNA vaccine. Therefore, the fusion gene can be used as a new potential candidate antigen in the development of subunit vaccine of Brucella.