Prokaryotic expression and in vitro activity of Ancylostoma ceylanicum calreticulin

Abstract Ancylostoma ceylanicum is a zoonotic soil-derived nematode that parasitizes the intestine of humans and animals (dogs and cats), causing malnutrition and iron-deficiency anemia of the host. Studies have shown that the parasite can regulate or block the host's immune response by secreting calreticulin. However, no data are available on A. ceylanicum calreticulin. To study the biological function of A. ceylanicum calreticulin (Ace-CRT), we amplified the cDNA encoding Ace-CRT and constructed the prokaryotic expression vector pET28-Ace-CRT to express the target protein. The antigenicity of Ace-CRT was investigated by western blotting with canine serum. Mouse splenocytes and canine peripheral blood lymphocytes were stimulated with the recombinant protein in vitro to explore its proliferation activity and effect on the transcription level of cytokines. The recombinant protein was co-incubated with sensitized sheep erythrocytes to analyze the inhibitory effect on the complement-mediated hemolysis. Results showed that the rAce-CRT was abundantly expressed in Escherichia coli, with good antigenicity. The recombinant protein stimulated the proliferation of mouse splenocytes and canine peripheral blood mononuclear cells (PBMCs). Quantitative reverse-transcription polymerase chain reaction showed that rAce-CRT mainly promoted the expression of Th2 cytokines, especially IL-13, in canine peripheral blood lymphocytes. In vitro, rAce-CRT inhibited complement-mediated sheep erythrocytes hemolysis. These findings indicate that Ace-CRT has an immunomodulatory role and might be a promising candidate molecule for the hookworm vaccine.