Expression and biological functions of Ancylostoma ceylanicum saposin-like protein

Abstract

Ancylostoma ceylanicum is a common zoonotic nematode that inhabits the small intestine of humans, dogs, and cats. Saposin-like proteins (SLPs) have hemolytic and antibacterial activities and could be used as diagnostic or vaccine candidates. To explore the biological functions of Ancylostoma ceylanicum SLP (Ace-SLP-1), cDNA-encoding Ace-SLP-1 mature peptide was cloned into prokaryotic expression vector pET-28a and transformed into Escherichia coli BL21 (DE3) to induce expression. After incubation of canine red blood cell suspension with different concentrations of recombinant Ace-SLP-1, the supernatant was separated to measure OD value and calculate the hemolysis rate. The different concentrations of recombinant protein were co-cultured with E. coli and Enterococcus faecalis, and colony-forming units (CFU) were determined by the plate counting method. Peripheral blood mononuclear cells (PBMCs) from healthy dogs were incubated with different concentrations of recombinant Ace-SLP-1, and the cytokine expression was evaluated by relative quantitative PCR. Our results showed that the hemolytic activity of Ace-SLP-1 increased with the increase in protein concentration from 25 to 100 μg/mL. The recombinant protein had no antibacterial activity against the two kinds of bacteria but could stimulate the secretion of cytokines (IL-4, IL-10, IL-12, and IL-13) in canine PBMCs. These data suggest that Ace-SLP-1 is involved in hookworm blood-feeding and survival and has good immunogenicity, supporting its potential as a diagnostic and vaccine target molecule.

Keywords: Ancylostoma ceylanicum; Antibacterial activity; Cytokine expression; Hemolytic activity; Saposin-like proteins.