Molecular characterization and expression of a cysteine protease from Clonorchis sinensis and its application for serodiagnosis of clonorchiasis

Abstract

Cysteine proteases play essential roles in parasite physiology as well as in host-parasite interactions through their modulation of various biological and pathobiological events. In the present study, a full-length sequence encoding cysteine protease of Clonorchis sinensis (CsCP) was isolated from our adult cDNA library. The open reading frame contains 984 bp encoding 327 amino acids. The present amino acid sequence shared 68% identity with two known CsCP genes and 29-49% identity with that of other species. Bioinformatics analysis showed that conserved domains and characteristic amino acid residues of cysteine proteases were observed in this sequence. Real-time PCR experiments revealed that CsCP was consecutively transcribed in various developmental stages of the parasite, including adult worm, excysted juvenile, metacercaria and egg. Recombinant CsCP (rCsCP) could be probed by rat anti-CsCP serum, rabbit anti-excretory-secretory products (ESP) serum and serum from human infected with Clonorchis sinensis in Western blot. The result of immunolocalization showed that CsCP was mainly located in the oral sucker, excretory bladder and tegument of cercariae and metacercariae, as well as the intestine of adult worm. The rCsCP-based IgG and its isotypes were all detected in sera from human infected with C. sinensis by enzyme-linked immunosorbent assay, and the level of IgG1 is the highest. The receiver-operating characteristic (ROC) analysis was used to determine the most appropriate cut-off value that yielded the high sensitivity (86.96%) and specificity (70.42%). These results revealed that CsCP may play an important role in the biology of C. sinensis and could be a diagnostic candidate for clonorchiasis.

The immunological characteristics and probiotic function of recombinant Bacillus subtilis spore expressing Clonorchis sinensis cysteine protease

“…The recombinant plasmid was transformed into E. coli BL21 (DE3) (BL21-pET28a- Cs CP, abbreviated as BL21- Cs CP) and induced by isopropyl β-D-thiogalactopyranoside (IPTG) as described previously [19]. The induced E. coli was collected by centrifugation and ultrasonicated to obtain inclusion bodies in sediment.…”

“…The cysteine protease superfamily of parasite organisms plays a key role in physiology and related pathobiology processes that are closely related to larval migration, nutrition acquisition, egg hatching and immune evasion [3, 17–19]. Currently, cysteine proteases identified from various parasites (e.g.…”

“…Currently, cysteine proteases identified from various parasites (e.g. C. sinensis , Fasciola hepatica , Taenia solium and Ancylostoma caninum ) have been exploited as major potential molecules for serodiagnosis, immuno-/chemo-therapy and vaccine candidates [19–25]. The cysteine protease from C. sinensis ( Cs CP) is an important component of excretory-secretory products (ESPs) in both adult worms and metacercariae [19, 26].…”

“…C. sinensis , Fasciola hepatica , Taenia solium and Ancylostoma caninum ) have been exploited as major potential molecules for serodiagnosis, immuno-/chemo-therapy and vaccine candidates [19–25]. The cysteine protease from C. sinensis ( Cs CP) is an important component of excretory-secretory products (ESPs) in both adult worms and metacercariae [19, 26]. Additionally, Cs CP is an indispensable protease participating in the excystment of metacercariae, enabling larvae to successfully move to bile ducts, and further develop into adults [26].…”

Investigation on oxidative stress of nitric oxide synthase interacting protein from Clonorchis sinensis

“…Clonorchiasis is caused by taking raw or undercooked freshwater fish containing infectious metacercariae (Lv et al 2011;Wang et al 2012;Rim et al 2005). Because of the high correlation between clonorchiasis and cholangiocarcinoma, C. sinensis infection has been considered as one of the most important biological agents of cholangiocarcinoma (Huang et al 2012;Young et al 2010;Bouvard et al 2009).…”