Abstract: Objective To characterize the biological function of calmodulin (CaM) from Clonorchis sinensis (C. sinensis, Cs) and
investigate its role in clonorchiasis-associated hepatic fibrosis. Methods The full-length sequence of CsCaM gene was isolated
from Cs cDNA library and its homologues were searched using BLASTx for comparison. Bioinformatics analysis was
performed to compare the homologues and predict the physiochemical characteristics and functional domains. The gene was
cloned in a prokaryotic plasmid and expressed in E. coli, and the recombinant protein was purified by affinity chromatography
for immunizing rats to produce polyclonal antibodies, whose titer was determined using ELISA analysis. Immunoblotting
analysis was carried out to determine of the purity and antibody recognition of CsCaM. Immunofluorescence assay was
employed to analyze the tissue location of the protein. A rat model of liver fibrosis was established by introperitoneal injection
of the recombinant protein. Results The recombinant CsCaM protein obtained contained 150 amino acids with a theoretical
molecular mass of 23.4 kD. CsCaM homologue had EF hand motifs. The recombinant pET-30a-CsCaM plasmid expressed in
BL21 E. coli was about 23.4 kD. The total IgG antibody titer in the immunized mice reached the peak level (over 1: 51200) 2 to
4 weeks after the first injection. Immunohistochemistry showed that CsCaM located in the testis of adult C. sinensis. The rats
receiving intraperitoneal injection of CsCaM showed severe liver inflammation with mild to moderate liver fibrosis.
Conclusion The pro-inflammation and pro-fibrosis effects of CsCaM in rat liver suggest its involvement in clonorchiasisassociated
hepatic fibrosis.