| Erohemorrhagic Escherichia coli O157:H7 is a highly pathogenic zoonnotic pathogen which is spread by food and water. Since the first separation, there have been different scales of breakout and sporadic epidemic in all parts of the world, caused significant economic losses to the breeding industry, and threatened the healthy of human beings constantly. Due to the different degree of infection, Erohemorrhagic Escherichia coli O157:H7 could cause bloody diarrhea, hemorrhagic colitis, hemolytic uremic syndrome and thrombocytopaenic purpura and even death. Who have listed it as emerging infectious disease with AIDS and Ebola hemorrhagic fever. The flagellin and C-Lysozyme inhibitor of Erohemorrhagic Escherichia coli O157:H7 play an important role in direct adherence and colonization.In this study, the secondary structures, hydrophilicity plot, flexibility plot, surface probability plot and antigenic index of the Flic gene and Ivy gene of Escherichia coli O157:H7 were analyzed by bioinformatics, and then chosed the potential B cell epitope peptides and were synthesized by chemical synthesis:Flic 338-349 KAASEGSDGASL、 Flic460-471 ATDTNKDYAPAI、Ivy 146-157 SLENHPDGFNFK. The polyclonal antibody was obtained by emulsifying the peptide in mice. ELISA showed that the titer of Anti-Flic-338-349, Anti-Flic460-471, Anti-Ivyl46-157 and the whole bacteria group was about 1:1600、1:6400、1:6400、1:12800. Western blot showed that the antibody could bind to the natural proteins specially. Indirect immunofluorescence test showed green fluorescence mainly in the action site of Hela cell and bacterial, which Flic338-349、Flic460-471 outside the cell, Ivy146-157 was in the protoplasm, the whole bacterial was both in the cell and outside. And the flyorescenec intensity value of immunity groups was 2 to 4 time to the normal control group that showed the great reactivity of the antibody.When the polyclonal antibody (Anti-Flic338-349, Anti-Flic460-471, Anti-Ivy146-157, Anti-EHEC O157:H7) was mixed with the Escherichia coli 0157:H7, the ability of Escherichia coli 0157:H7 adhere to HeLa cells weakened or lost, fluorescence intensity decreased 2-3 times compared to the group with no pretreatment, with significant difference, indicating that the polyclonal antibody can be combined with Escherichia coli O157:H7 natural protein, in vitro successfully blocked the adhesion of Escherichia coli O157:H7 in HeLa cells.After 3 times immunization, mice of each group were infected with this strains (LD50) by oral, bacteria could found in all mice. Data of the fecal excretion in the immunized groups was significantly lower than that in the non immune group. The protection rate of mice in Flic338-34、Ivyl46-157、Flic460-471 and the whole bacteria group was 80%、90%、100%、 100%, while the infection group was 30%, and the tite of Anti-Flic-460-471, Anti-Flic338-349, Anti-Ivy 146-157 and the whole bacteria group was 1:12800、1:1600、 1:12800、1:51200. Those results showed a good protection in infection, the antibody could neutralize with the Ecoli O157:H7 in vivo.During the experiment, the lymphocyte proliferation was obvious, the levels of IL-2、 IL-6、IL-12、IFN-y、TNF-a and mRNA expression levels were significantly increased. These data suggested the cellular immunity was induced by the polypeptide.In this study, results showed that the obtained antibody stimulated by the peptides Flic460-471、Flic338-349、Ivy 146-157 could recognize the native proteins, successfully inhibited the adhesion of Escherichia coli O157:H7 in Hela cells, proved protection to the mice in infected, and induced the secretion of IL-2、IL-6、IL-12、IFN-γ、TNF-a. Those data confirmed the peptides have the ability to stimulate the body to produce humoral and cellular immune responses. The peptides Flic460-4711、 Flic338-349、Ivy146-157 is the potential B cell epitope peptides of flagellin and lysozyme inhibitor of Escherichia coli O157:H7. |
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