Expression of a tick toxin for the development of a canine vaccine

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Acute, ascending, flaccid motor paralysis of forelimbs and death due to respiratory failure is the dominant characteristic of tick toxicosis by the Australian paralysis tick, Ixodes holocyclus. A tick toxin vaccine has the potential to be an effective preventative measure against tick toxicosis that affects thousands of domestic and companion animals each year. In previous research for the development of an anti-tick vaccine, Masina (1999) used a maltose-binding protein (MBP) holocyclus toxin (HT-1) fusion protein as immunogen. It was partially protective against challenges with crude tick extract in neonatal mice but unable to protect dogs against paralysis caused by direct tick attachment. Subsequently, four monoclonal antibodies generated in this study against the fusion protein were found to be incapable of binding to the native toxin in crude tick extract in Western blots. These results indicated that the HT-1 component of the MBP fusion protein was incorrectly folded compared to the native toxin. An expression system using ubiquitin fusion protein was investigated. Ubiquitin is a 10 kDa carrier protein which is smaller than the 43 kDa MBP. A smaller fusion partner was considered as it is less likely to interfere with HT-1 folding. The ubiquitin-HT-1 fusion protein was expressed in both soluble and insoluble forms, corresponding to the cytoplasmic fraction and inclusion bodies. The soluble form could be purified under non-denaturing conditions utilising the incorporated His-6-tag and a Ni affinity column. The insoluble form, like the periplasmically expressed MBP-HT-1 fusion protein, could only be purified using denaturing conditions. The purified soluble ubiquitin-HT-1 fusion protein appeared to have the correct conformational folding as it was recognized by commercial dog anti-tick serum in Western blots. The purified soluble ubiquitin-HT-1 fusion protein was used to immunise rabbits and mice for protection experiments, but was found to be unprotective. However, serum from immunized animals was able to detect a 5 kDa protein from crude tick extract in a Western blot. This 5 kDa protein was also recognized by the commercial dog anti-tick serum and has the molecular weight corresponding to the HT-1 neurotoxin. Creation of monoclonal antibodies to this ubiquitin-HT-1 fusion protein may therefore aid future development towards a tick vaccine. These antibodies could in turn be used to isolate native HT-1 from the crude tick extract instead of a combination of conventional chromatography methods. This approach would allow isolation of HT-1 in more significant quantities than is currently possible to enable confirmation of the HT-1 sequence. Suspected problems associated with incorrect sequence or interference by the carrier protein to cause a non-protective response could also be resolved.
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