Characterization of Hlcyst-3 as a member of cystatins from the tick Haemaphysalis longicornis.

The gene encoding cystatin from the tick Haemaphysalis longicornis has been reported previously. In the study reported here, we characterized a member of cystatins and designated it as Hlcyst-3 (H. longicornis cystatin-3). Its full-length cDNA is 602 bp, and it encodes a putative 129 amino acid protein with an obvious signal peptide. Sequence analysis revealed that it has significant homology with the known secreted cystatin. The recombinant protein was expressed in a GST-fused soluble form in Escherichia coli and was purified by affinity chromatography. The inhibitory activity of the recombinant protein against papain and cathepsin L was identified by fluorogenic substrate analysis. Real-time PCR revealed that Hlcyst-3 was mostly expressed in the tick midgut.