Infectious diarrhoea remains an emerging problem in the world health program. Among diarrheagenic agents, Vibrio cholerae and enterotoxigenic and enterohemorrhagic Escherichia coli are critical enteropathogens. AB5 toxin produced by these bacteria, heat-labile enterotoxin (LT), cholera enterotoxin (CT), and shiga-like cytotoxin (STX) can target the immune system and are subunit vaccine candidates. A chemically-synthesized chimeric construct composed of the binding subunits of these toxins (LTB, STXB, and CTXB) was developed based on bioinformatics studies. The whole chimeric protein (rLSC) and each of the segments (rLTB, rSTXB, and rCTXB) were expressed in a prokaryotic expression system (E. coli), purified, and analysed for their immunogenic properties. The results indicate that these recombinant proteins were effectively able to present appropriate epitopes to an animal model of the immune system which could result in and increase IgG in serum and immune responses that protect against the binding activity of these toxins. The immunological assays revealed that the sera of immunized mice prevented toxins from binding to their specific receptors and neutralized their toxic effects. The proposed construct should be considered as a potent immunogen to prevent toxicity and diarrhoea.
Background
Newcastle disease virus (NDV) is a dangerous viral disease, infecting a broad range of birds, and has a fatal effect on the poultry industries. The attachment and consequently fusion of the virus to the host cell membrane is directed by the two superficial glycoproteins, the hemagglutinin-neuraminidase (HN) and the fusion (F) which is considered as the important targets for the poultry immune response.
Objectives
The principal goal of this investigation was to realize the potential efficacy of the
E. coli
expression system for the production of the multi-epitopic HN, and F proteins with respect to the ability for the stimulation of the immune system and production of the cross-reactive antibodies in mice.
Materials and Methods
The recombinant HN and F (rHN, rF) have accumulated almost 40% of the total bacterial proteins. The presence of rHN and rF proteins recognized by the Western blotting with specific anti-HN, anti-F, anti-Newcastle B1, and anti-poly 6x His-tag antibodies. Furthermore, both rHN and rF have shown the specific reactivity against the Newcastle B1 antiserum as a standard strain.
Results
The ELISA analysis showed that the higher dilutions of the antibody against Newcastle B1 could react with the as least quantity as 100 ng of the purified rHN, and rF. Cross-reactivity analysis of the sera from the mice immunized with Newcastle B1 in two time points indicated that the raise of anti-Newcastle B1, anti-HN and anti-F antibodies peaked at 28 days post immunization (dpi). Moreover, temporal variation in IgG titration between both time points was significant at 5% probability level.
Conclusion
The results provided valuable information about the cross-reactivity patterns and biological activity of the multi-epitopic proteins compared to the NDV standard strain which was determined by the Western blotting and ELISA.
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