Aspartyl protease inhibitors (APIs) from parasitic intestinal nematodes are highly immunogenic and have been suggested as potential vaccine antigens. Ac-API-1 from Ancylostoma caninum showed strong immunogenicity and its polyclonal antibodies could specifically recognize the excretory/secretory products of adult worms. However, little is known about molecular characteristics and biological function of API from Ancylostoma ceylanicum (Ace-API). In this study, the Ace-API mature peptide coding sequence was cloned and expressed, and molecular characteristics of its full length sequence were analyzed. Ace-API cDNA was 684 bp in length, which encoded 228 amino acids. The similarity of the Ace-API amino acid sequence to Ac-API-1 and Adu-API-1 was 96.93% and 96.49%, respectively, and they clustered together in the phylogenetic tree. Escheria coli-expressed recombinant protein was mainly soluble in the supernatant of bacterial cell lysate. Western blot showed that Ace-API protein had good reactivity to the serum of infected dogs. Pepsin inhibition assay revealed that the recombinant protein had inhibitory activity on pepsin. Immunofluorescence results demonstrated that Ace-API was mainly localized to the epidermis, excretory glands, and pseudocoelomic fluid of the adult. Using the quantitative real-time PCR, the expression of Ace-api mRNA in adults was significantly higher than that in the third stage (L3) larvae. Together, these data indicate that Ace-API is secreted extracellularly by the parasite, and might play a role in protecting the parasite against the proteolytic digestion by the host proteases, which stimulate further studies to explore this protein as a potential hookworm vaccine candidate.
Keywords: Ancylostoma ceylanicum; Aspartyl protease inhibitor; Differential expression; Immunolocalization; Inhibitory activity.
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