Identification and characterization of the cysteine protease inhibitor gene MdCPI from Musca domestica

Cysteine proteinase inhibitors (CPIs) are involved in many vital cellular processes such as signalling pathways, apoptosis, immune response and development; however, no CPIs have yet been reported from the housefly Musca domestica. Here we report the isolation and characterization of a housefly CPI gene designated MdCPI. The gene contains an open reading frame of 357 bp encoding a protein of 118 amino acid residues with a putative signal peptide of 17 amino acid residues. Protein alignment demonstrated a high homology to that of Sarcophaga crassipalpis (identity = 51%). Phylogenetic analysis suggested that all CPIs from dipterans, including the housefly, belong to the I25A family and may be descended from a single common ancestor. The gene was expressed in and purified from Escherichia coli. Biochemical studies showed that MdCPI exerts an inhibiting function on papain, which is a classical assay to confirm CPIs. Real-time quantitative PCR and immunolocalization analysis revealed that MdCPI is specifically expressed in haemocytes and fat bodies. It is highly down-regulated in larvae and markedly up-regulated in the pupal stage, suggesting that it may be related to development.