For

immunoblotting, proteins were separated by SDS–PAGE and the gels were electroblotted onto a PVDF membrane (Pall Corporation, East Hills, NY). Anti-rHp-CPI mAb was used as the primary antibody, and horseradish peroxidase-conjugated anti-mouse IgG (Thermo Fisher Scientific, Guangzhou, China) diluted 1 : 50 000 was used as the secondary antibody. Bound antibody was detected using enhanced chemiluminescence reagents (Thermo Fisher Scientific). Statistical analyses were performed with GraphPad Prism 5 software (GraphPad Software Inc., La Jolla, CA). Significance of differences between groups was analysed using the Student’s t-test. Data are presented as mean ± SD. A P-value < 0·05 was considered significant. Cystatin is known to be conserved in eukaryotes and has Ribociclib manufacturer been identified in many species of nematode parasite.[18] To determine if the H. polygyrus parasite has the CPI gene, we screened the cDNA library of H. polygyrus by RT-PCR using the primers for consensus sequences of cystatin reported in other nematode parasites, and obtained a fragment of cystatin. We then obtained the full length of CPI gene from H. polygyrus (Hp-CPI) using the RACE technique. The open reading frame of Hp-CPI has 432 bp, and the cloned

protein (rHp-CPI) consisted of 143 amino acids (Fig. 1a). Comparison of the Hp-CPI amino acid sequence with cystatins from other nematodes showed various levels of homology (Fig. 1a). Epigenetics inhibitor As observed in cystatin from other nematode species, the CPI protein from H. polygyrus contains a signal peptide of 21 amino acids indicating that the Hp-CPI is a secreted protein. In immunoblotting assay, we confirmed that the mAb generated against the rHp-CPI was able to react with a protein component of 14 000 molecular weight from the excretory and secretory products

prepared from adult H. polygyrus (Fig. 1b). We then examined the protease inhibitory ability of rHp-CPI to confirm the biological activity of the rHp-CPI protein. The rHp-CPI was produced in E. coli, affinity-purified and analysed for its ability to inhibit the proteolytic activity of cathepsin B, C, L and S, which are known to be important in functions Tacrolimus (FK506) of antigen presentation cells.[30, 31] We observed that rHp-CPI inhibited the proteolytic activities of cathepsin B, C, L and S in a dose-dependent manner (Fig. 2a), indicating that the recombinant CPI protein from H. polygyrus possesses the biological function of protease inhibition activity. We also analysed the protease inhibitory activity of the ES products from H. polygyrus and observed that H. polygyrus ES products were able to inhibit the proteolytic activities of cathepsin B, C, L and S (Fig. 2b). Although H.