Title:
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Analysis of the Brown Dog Tick, Rhipicephalus sanguineus, salivary gland transcriptome for potential drug targets
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The dopamine receptor is critical in fluid secretion from tick salivary glands and, as such, was perceived as a potential drug target. Homology cloning was employed to isolate a dopamine receptor from R. sanguineus salivary glands. A dopamine receptor was successfully cloned from locust salivary glands although not from R. sanguineus salivary glands. A cDNA library was constructed from feeding female R. sanguineus salivary glands and random mass sequencing of 1440 clones was performed. Sequences were identified from database searches and classified according to gene ontology. Several enzymes were identified as potential drug targets and a glutathione S-transferase (GST) was chosen for further study. The dopamine receptor was not identified, as were no other G protein-coupled receptors. A mu class GST from R. sanguineus salivary glands was expressed as a recombinant protein in E. coli and was found to have differences when compared to other tick GSTs, namely low catalytic activity with a model substrate. Preliminary studies suggested that the GST is not involved in prostaglandin synthesis and the GST bound haematin with high affinity. Of several tissues investigated within female ticks, the GST mRNA was only expressed in the salivary glands and only in glands from feeding female ticks indicating a potential role in blood feeding. Two approaches were employed to identify drug targets in R. sanguineus salivary glands. It is concluded that whilst random mass sequencing of a cDNA library yields many potential targets, because of the nature of the salivary gland tissue with high repetition of mitochondrial and secretory protein RNA, homology cloning would be the recommended approach for scientific, low abundance targets.
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