The sequence expression and immunological characterisation of the HT-1 neurotoxin from the Australian paralysis tick Ixodes holocyclus
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The paralysis tick of Australia, Ixodes holocyclus, causes a severe toxicosis in domestic animals such as dogs and cats, livestock, and in some cases humans. It is characterised by a rapidly ascending flaccid paralysis. The causative agent of the toxicosis is a neurotoxin produced in the tick salivary glands. The current treatment for tick paralysis is in the form of a polyclonal dog antiserum. This antiserum treatment is expensive and effective only in the early stages of paralysis. This thesis describes the isolation of the cDNA sequence for the neurotoxin HT-1 of I. holocyclus. In addition it describes the successful expression of the mature HT-1 toxin as a fusion protein in a bacterial host system and attempts at expression of this recombinant protein in the absence of a fusion partner in a baculovirus system. The antigenic and protective properties of the HT -1 fusion protein raised in mice and dogs is also discussed. The complete cDNA sequence of the HT-1 neurotoxin was determined using the technique of RACE-PCR. The derived protein sequence of the mature toxin has a calculated molecular weight of 5.9 kDa and contains 8 cysteine residues, suggesting that it can form four disulphide bonds. The HT-1 protein sequence shares similarities with other arachnid neurotoxins. Examples of these similarities are its small size, number and arrangement of cysteine residues and in the length and composition of its signal sequence. A recombinant form of the HT-1 toxin was produced as a fusion protein in an E.coli host system. The fusion protein was shown to be immunogenic by Western blot and ELISA analysis. The antibodies produced against the recombinant fusion protein show partial protection against native toxin in mouse assays. Dogs immunised with the recombinant fusion protein produced specific antibodies to HT-1 as demonstrated in ELISA assay. However, these antibodies were not protective when the dogs were challenged with numerous live ticks. The results obtained have contributed a novel tick neurotoxin sequence which as a fusion protein produces antibodies that exhibit partial protection in mice against native toxin challenge. These results provide some optimism and basic understanding of what is required for the future development of a recombinant vaccine against paralysis caused by the tick I. holocyclus.
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