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This article is part of the series Aspects of parasitology in the People's Republic of China: surveillance, diagnosis, infection and immunity.

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Molecular characterization of cathepsin B from Clonorchis sinensis excretory/secretory products and assessment of its potential for serodiagnosis of clonorchiasis

Wenjun Chen12, Xiaoyun Wang12, Xuerong Li12, Xiaoli Lv12, Chenhui Zhou123, Chuanhuan Deng12, Huali Lei12, Jingtao Men12, Yongxiu Fan12, Chi Liang12 and Xinbing Yu12*

Author Affiliations

1 Department of Parasitology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou 510080, People's Republic of China

2 Key Laboratory for Tropical Diseases Control, Sun Yat-sen University, Ministry of Education, Guangzhou 510080, People's Republic of China

3 Department of Gynecology and Pediatrics, Nursing School, Guangdong Medical College, Dongguan, 523808, PR China

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Parasites & Vectors 2011, 4:149  doi:10.1186/1756-3305-4-149

The electronic version of this article is the complete one and can be found online at:

Received:23 March 2011
Accepted:27 July 2011
Published:27 July 2011

© 2011 Chen et al; licensee BioMed Central Ltd.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.



Cathepsin cysteine proteases play multiple roles in the life cycle of parasites such as food uptake, immune invasion and pathogenesis, making them valuable targets for diagnostic assays, vaccines and drugs. The purpose of this study was to identify a cathepsin B of Clonorchis sinensis (CsCB) and to investigate its diagnostic value for human helminthiases.


The predicted amino acid sequence of the cathepsin B of C. sinensis shared 63%, 52%, 50% identity with that of Schistosoma japonicum, Homo sapiens and Fasciola hepatica, respectively. Sequence encoding proenzyme of CsCB was overexpressed in Escherichia coli. Reverse transcription PCR experiments revealed that CsCB transcribed in both adult worm and metacercaria of C. sinensis. CsCB was identified as a C. sinensis excretory/secretory product by immunoblot assay, which was consistent with immunohistochemical localization showing that CsCB was especially expressed in the intestine of C. sinensis adults. Both ELISA and western blotting analysis showed recombinant CsCB could react with human sera from clonorchiasis and other helminthiases.


Our findings revealed that secreted CsCB may play an important role in the biology of C. sinensis and could be a diagnostic candidate for helminthiases.


Clonorchis sinensis is the causative agent of clonorchiasis, a chronic liver infection of human acquired through consumption of raw or undercooked fish and shrimps with infectious metacercariae. Clonorchiasis is endemic in Asian countries and over 35 million people globally are infected C. sinensis, including an estimated 15 million in People's Republic of China [1]. Recently, this infection has emerged in non-endemic regions and developed countries following growing international markets, improved transportation systems and demographic changes such as population movements [2]. C. sinensis adults reside chronically in the biliary tract and cause periductal inflammation, fibrosis, pyogenic cholangitis, biliary calculi, cholecystitis, liver cirrhosis and pancreatitis [3]. Like Opisthorchis viverrini, C. sinensis is one of the direct causes of cholangiocarcinoma announced by the International Agency for Research on Cancer (IARC) in 2009 [4]. It is important to take some measures to control clonorchiasis due to its public health threat. Until now, the main prevention and control strategies for this parasite are treatment of individual patients with praziquantel, and interrupting transmission at the intermediate host level [5]. However, there have been little effective measures to prevent this neglected tropical disease [6].

Cysteine proteinase is ubiquitous in all species [7-9]. In parasites, cysteine proteases have attracted much attention for their essential roles in parasite physiology as well as in host-parasite interactions through their modulation of various pathobiological events, including host tissue invasion, nutrient uptake, host immune evasion and molting [10-13]. Research has been conducted to characterize the biochemical properties and pathophysiological roles of cysteine proteases from trematode parasites. The essential roles of cysteine proteases in parasite survival or growth make them attractive targets for vaccines or chemotherapeutic agents [14-16]. Several genes encoding C. sinensis cysteine proteases have been identified and partially characterized [17-19]. Lee et al. [20] reported that cathepsin F-like cysteine protease of C. sinensis is a good vaccine candidate against clonorchiasis. Li et al. [21] found that endogenous cysteine proteases of C. sinensis metacercariae are probably involved in the excystment process. Kang et al. [22] indicated that partially purified cysteine protease from excretory/secretory products (ESP) of C. sinensis adults exhibited significant cytotoxic effects against cultured cells. ESP of parasites have attracted more attention for their significant roles in the diagnosis, vaccine, drug target and host-parasite interactions etc. In vitro biochemical studies have predicted that ESP from liver flukes have definitive roles in feeding behavior, detoxification of bile components and immune evasion [23]. Ju et al. [18] have identified legumain from ESP as a serodiagnostic antigen of clonorchiasis. In addition, several genes encoding C. sinensis cysteine proteases have also been identified and their value as diagnostic antigens for clonorchiasis was investigated [24,25]. However, little is known about cathepsin B (CB) in C. sinensis except five distinct sequences deposited in Genbank.

As members of the cysteine protease family, cathepsins have been assayed in the serodiagnosis of both human and animal in parasite infections. Cornelissen et al. [26] reported a specificity of 75.3% in naturally infected cattle using Fasciola hepatica cathepsin L as coating antigen. Carnevale et al. [27] found that recombinant pro-cathepsin-L was 100% specific in the diagnosis of human F. hepatica infection. Sripa et al. [28] indicated that Ov-CB-1 was acceptable in ELISA for the serodiagnosis of human opisthorchiasis with 67% and 81% of sensitivity and specificity, respectively. To find out whether CB could be applied for serodiagnosis in C. sinensis infection, we identified a gene encoding cathepsin B of C. sinensis (CsCB) and investigated its diagnostic value for human helminthiases.


Sequence analysis of CsCB gene sequence

The complete coding sequence of CsCB is comprised of 1,020 bp encoding a putative protein of 339 amino acids with a predicted molecular mass of 37.9 kDa and an isoelectric point of 5.32. The ORF consists of a hydrophobic signal peptide at the N-terminus, followed by a pro-region of between 70 and 71 AA and a mature protease sequence. BLASTx showed that it shared 63%, 52%, 50% and 53% identity with CB of Schistosoma japonicum, Homo sapiens, F. hepatica and Echinococcus multilocularis, respectively. Highly conserved residues of the catalytic triad (Cys116, His285 and Asn305) as well as the Gln could be searched in this sequence. The putative protein contained an occluding loop that is the signature of cathepsin Bs and a haemoglobinase motif which is shared by helminth blood-feeders (Figure 1).

thumbnailFigure 1. Alignment of cathepsin B (CsCB) deduced amino acid sequence from C. sinensis and other species. The deduced amino acid sequence shares 63%, 52%, 50% and 53% identity with CB of S. japonicum, H. sapiens, F. hepatica and E. multilocularis, respectively. Highly conserved residues are shaded in gray. Residues of the catalytic triad (Cys116, His285 and Asn305) are indicated with black triangles. The oxyanion Gln is marked with white triangle; arrow shows cleavage point of mature enzyme, the occluding residues and hemoglobinase motif are boxed.

Cloning, expression and purification of recombinant CsCB (rCsCB) in E. coli

The recombinant pET-28a(+) plasmid containing the CB gene coding region (signal peptide removed) was confirmed by digestion with restriction enzyme. DNA sequencing revealed that the construct was correct with 6 × His tag at the N terminus of the recombinant protein. The recombinant CsCB was expressed in inclusion bodies in E. coli. SDS-PAGE showed that the molecular mass of fusion protein was about 40 kDa, which was in correspondence with the predicted 39.2 kDa (including 34 amino acids of vectors). After purification and renaturation, the concentration of the recombinant protein was about 0.25 mg/ml (Figure 2A).

thumbnailFigure 2. Expression, purification and identification of CsCB. A. Expression and purification of CsCB by 12% SDS-PAGE. M, protein molecular weight markers; lane 1, lysate of E. coli with pET-28a(+) without IPTG induction; lane 2, lysate of E. coli with pET-28a(+) with IPTG induction, lane 3, lysate of E. coli with pET28a(+)-CsCB before induction; lane 4, lysate of E. coli with pET28a(+)-CsCB after induction; lane 5, supernatant of induced pET28a(+)-CsCB; lane 6, sediment of induced pET28a(+)-CsCB; lane 7, purified CsCB. B. Western blotting analysis of recombinant CsCB. Lane 1, recombinant CsCB reacted with the sera from normal rats; lane 2, recombinant CsCB reacted with the sera from rats immunized with recombinant CsCB; lane 3, recombinant CsCB reacted with the sera from C. sinensis-infected rats; lane 4, recombinant CsCB reacted with the sera from rats immunized with the ESP.

Identification of CsCB as ESP by western blotting

Rat anti-rCsCB antibody titers were higher than 1:102,400 as determined by ELISA. In western blotting assay, both C. sinensis-infected rat serum and anti-CsCB rat serum could react with the CsCB while the normal rat serum could not. Comparing with the control group, sera from rats immunized with total ESP could recognize CsCB. In addition, ESP has been shown to react with anti-CsCB rat serum (data not shown). The results above indicated that CsCB was a component of ESP (Figure 2B).

RT-PCR analysis of CsCB at life-stage of C.sinensis

CsCB transcrips were detected both in adults and metacercaria of C. sinensis (Figure 3A), and the expression level in adults was higher than that of metacercaria when normalized by β-actin (Figure 3B, P < 0.05).

thumbnailFigure 3. Transcriptional level of C. sinensis CB at metacercaria and adult worm. A. 1% agarose gel. DNA marker (M), CsCB PCR products amplified from adult worm cDNA (lane 1), metacercaria cDNA (lane 2), β-actin amplified from adult worm cDNA (lane 3), metacercaria cDNA (lane 4) and CsCB PCR product amplified from recombinant CB plasmid (lane 5). B. PCR products were quantified and analyzed. PCR products were quantified by Tanon Gis software, compared normalized by C. sinensis β-actin, analyzed by Student's t test (Software package SPSS16.0). P-value of < 0.05 indicated statistical significance.

Immunohistochemical localization of CsCB in C. sinensis adults

Using the antisera against CsCB as the primary antibody and fluorescence labeling IgG as the secondary antibody, immunolocalization showed that CsCB distributed in the intestine of adults, while no specific fluorescence except autofluorescence was detected in adults treated with normal serum (Figure 4).

thumbnailFigure 4. Immunolocalization of CsCB in adult worm of C. sinensis. Rat anti-CsCB serum was used as primary antibody and goat anti-rat IgG as the secondary antibody. Panel A and D show tissues of adult worm under fluorescence microscope. Panel A and C show the same part under white light. Panel A and B were the negative control treated with preimmune rat serum. i, intestine s, sucker. The images were magnified at 100× for photograph.

Serodiagnosis of human helminthiases

The optimal concentration of coating antigen and dilution of serum samples determined by checker board titration were 3 μg/ml and 1:400, respectively. The cut-off value for positive infection status in ELISA tests evaluated by receiver operating characteristic (ROC) curve was 0.47 (Figure 5). Sera from humans infected with C. sinensis showed a sensitivity of 79% by ELSA based on rCsCB and healthy sera and sera from those infected with other helminths showed a specificity of 81% (Table 1). Both ELISA and western blotting assays showed that rCsCB could react with human sera from clonorchiasis, fascioliasis, schistosomiasis, paragonimiasis, cysticercosis and echinococciosis (Figure 6).

thumbnailFigure 5. ROC analysis of performance of rCsCB as a serodiagnostic antigen. ROC curve of rCsCB employed as the antigen in ELISA, calculated from OD450 values of sera from parasitologically positive and negative clonorchiasis individuals. The curve was plotted between sensitivity and 1-specificity for different cut-off values of OD450. The curve showed that the optimal cut-off value at OD450 was 0.47 with the area under curve (AUC) was 0.84.

Table 1. Summary of difference between rCsCB ELISA and stool examinations results.

thumbnailFigure 6. Immune response to human helminthiases of rCsCB. 1-7: clonorchiasis, fascioliasis, schistosomiasis, paragonimiasis, echinococciosis, cysticercosis and healthy serum, respectively. A. Serodiagnosis of human helminthiases by ELISA(scatter plot). B. Immune reactionsshown by western blotting. The probed bands were indicated with arrow.


Cathepsins in general are of interest to parasitologists, as there is considerable evidence that they play a key role in the biology of parasites [29]. In this study, a CB of C. sinensis was cloned and overexpressed in E. coli. It was classified as CB due to its sequence homology to cathepsin B protein and structure. The putative amino acid sequence shared 63%, 52% and 50% identities with cathepsin B from S. japonicum, H. sapiens and F. hepatica, respectively. Sequence analysis showed that CsCB has typical catalytic residue of cysteine, histidine and asparagine, as well an occluding loop that is the signature of cathepsin Bs [30]. A haemoglobinase motif which is shared by helminth blood-feeders could be found in this deduced sequence [31]. Since C. sinensis generally feed on bile and epithelial cells rather than blood, however, it is thought that this motif may be an important tool for identifying potential hemoglobinases and contribute to haemoglobin degradation [32]. The occluding loop is a distinctive feature of CBs; it is not only responsible for exopeptidase activity to CBs, but also governing the pH dependence of auto-activation. In F. hepatica, Beckham [33] reported that recombinant FhCatB1 did not auto-activate upon secretion by yeast but could be auto-activated in a low pH buffer. As it stands, CsCB may develop auto-activation in an appropriate pH buffer, so further studies will be carried out to investigate the auto-activation and enzyme activity of this protease.

Cathepsin B proteases of parasitic helminths such as F. hepatica and Schistosoma mansoni have been well studied. In F. hepatica, for example, there are at least 10 genes encoding CB being identified by using proteomics and transcriptome analysis, among which six are components of ESP of immature flukes. RNAi directed to FhCB2 reduced invasion of F. hepatica newly excysted juveniles through the intestine in vitro and vaccine with F. hepatica CB2 reduced fluke burdens and liver damage in rats following challenge, revealing that FhCB2 was a key molecule in F. hepatica biology and drug therapy [34]. In S. mansoni, SmCB1 and SmCB2 have been identified and the function of SmCB1 was examined using RNAi silencing, which illustrated that SmCB1 played a role in nutrient acquisition [35,36]. Based on the key role of CB in the biology of parasites, we expect that CsCB may play the same role in nutrition intake and immune invasion of host. In multicellular parasites such as trematodes, the intestine is a major source of secreted proteases and also a place for nutrition digestion and absorption. According to results of immunolocalization, CsCB was distributed in the intestine of adult worm, indicating that CsCB might be involved in digestion of host protein and nutrient uptake for this parasite itself. In our RT-PCR experiments, CsCB could be detected in both adult worm and metacercaria of C. sinensis. As a secreted protease, these observations suggested that CsCB may play an important role in the biology of this parasite.

Proteases contained in ESP of parasites released to the environment, play key roles in bile duct malignancy and the subsequent development of cholangiocarcinoma [37,38]. Our previous studies revealed that ESP of adult C. sinensis lysophospholipase and phospholipase A2 might be pathogenic factors of human hepatic fibrosis caused by infection of C. sinensis [39,40], which could deepen our understanding of the pathogenesis of C. sinensis. CB has already been found in ESP of C. sinensis and some other helminths [41]. Moreover, western blotting revealed that CsCB was a component of ESP, and CsCB could react with C. sinensis-infected rat serum. In ELISA results, rats injected with recombinant CsCB developed high antibody titers. Our results showed that CsCB might be involved in the pathogenesis of C. sinensis-related hepatobiliary diseases as a component of ESP with antigenicity and immunogenicity. Indeed, C. sinensis adults dwell in the bile duct of host, persistently released ESP resulting in mechanical damage and chemical stimulus which must have brought on infiltration of inflammatory cells surrounding bile ducts and adenomatous hyperplasia of biliary epithelia [42-44]. However, there remains much to do to elucidate the mechanism of C. sinensis causing hepatobiliary diseases. We have also approached the antigenicity and diagnostic value of CsCB in human helminthiases. Both the ELISA and western blotting showed that CsCB could react with sera from most of human helminthiases, but not with sera from healthy people. High identity in the predicted amino acid sequences of C. sinensis and other helminths could be responsible for the observed cross-reactivity. Though cross-reactivity existed, CsCB could also be applied as a diagnostic candidate of clonorchiasis since treatment with praziquantel has the same efficacy on helminths.


A gene encoding cathepsin B protein of adult C. sinensis was cloned and expressed for the first time. Our findings revealed that CsCB, which is expressed in the intestine and released outside the worm as a component of ESP, may play an important role in the biology of C. sinensis. Moreover, CB may play conserved roles in helminths for the high homology analyzed by bioinformatics. Further investigations are required to characterize the conserved functions of such important protease and the role as a potential vaccine candidate against C. sinensis infection.

Materials and methods

Sequence analysis of CsCB gene sequence

A complete coding sequence encoding CB was isolated from GenBank (Accession no. EF102086.1) in NCBI webcite, the physicochemical properties and the functional domains in deduced amino acids were predicted by proteomics tools in ExPaSy web site webcite. Based on the similarity, the homologous sequences of different species, including E. multilocularis (BAJ83491.1), F. hepatica(ABU62925.1), S. japonicum (CAX71086.1), H. sapiens (NP_001899.1), Paragonimus westermani (AAB93494.1) and Taenia solium (AAS00027.1) were identified using the basic local alignment search tool (BLAST) server. Multiple sequences alignments were performed using bioinformatics analysis software Vector NTI suite 8.0.

Cloning, expression, purification and refolding of CsCB in E. coli

Gene sequence encoding CsCB (signal peptide excluded) was amplified from cDNA of C. sinensis by polymerase chain reaction (PCR) using forward primer: 5'-CGC

GAGTATATTCCATCTTTCGA-3' and the reverse primer: 5'-GTC
TCACAGTTTTGGATGACC-3' with BamHI/XhoI restriction enzyme sites (underlined). PCR was carried out for 30 cycles at 94°C for 45 s, 57°C for 45 s, and 72°C for 60 s, and the reaction continued for 10 min at 72°C after the last cycle. Purified PCR products were cloned into the His6 tag expression vector pET-28a(+) (Novagen; USA) with corresponding incision enzymes. The recombinant plasmid was transformed into E. coli for expression and insertion confirmed by digestion with restriction enzyme and DNA sequencing. Expression of recombinant CsCB protein was induced by isopropyl-β-D-thiogalactoside (IPTG) at a final concentration of 0.2 mM for 3 h at 30°C. The bacterial cells were collected by centrifugation at 4°C, and the inclusion bodies containing the recombinant fusion protein were solubilized completely with 6 M urea in 20 mM Tris-HCl buffer (pH 8.0), followed by purification with His Bind Purification kit (Novagen; USA) and elution with 150 mM imidazole. Renaturation was carried out by stepwise diluting urea in dialysate buffer (20 mM Tris-HCl, 5 mM EDTA buffer, pH 8.0). Purified protein was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and stained with Coomassie blue, final recombinant protein concentration was estimated by Bradford assay using BSA method used as a standard.

Semi-quantitative reverse transcription-PCR (RT-PCR) analysis of CsCB

In order to determine the mRNA transcriptional level of CsCB in various stages of the parasite, total RNA of adult worms and metacercariae were extracted and quantitated by nucleic acid/protein analyzer (Beckman Coulter; USA). Reverse transcription reactions were carried out by transforming equal amounts of total RNA (1 μg each) to cDNA using RT-PCR Kit (TaKaRa; PR China). RT-PCR experiments were employed to amplify the transcripts of CsCB from cDNA of adult worms and metacercariae, respectively, and C. sinensis β-actin (GenBank accession no. EU109284) was used as a positive control. The forward and reverse primer for CsCB were 5'-GGATTCGGCCTGGAAAAAC-3', 5'-CAGTTTTGGATGACCAGCAT-3' and for β-actin were 5'-GGTGACGCTGAAGTATCCTATTGA-3', 5'-CCAAAGCATAGCCCTCGTAGAT-3', respectively. The programs for PCR were the same as described above. PCR products from two life stages were quantified by Tanon Gis software (Tanon 4100; PR China) normalized by C. sinensis β-actin and analyzed by Student's t test (Software package SPSS16.0), P-value of < 0.05 indicated statistical significance.

Preparation for the antisera of recombinant protein and C. sinensis excretory/secretory product (ESP)

Living adults of C. sinensis were collected and cultured in RPMI-1640 (Gibco; USA) at 37°C under 5% CO2 for 6 h. The culture was centrifuged at 12,000× g at 4°C for 30 min to remove insoluble debris. The supernatant was dialyzed in PBS for 12 h and concentrated with sucrose. Six-week-old male Sprague-Dawley rats were purchased for animal experiments under the Guide for the Care and Use of Laboratory Animals. Both the recombinant CB and ESP were emulsified with complete Freund's adjuvant and immunized subcutaneously to SD rats. Each rat was given 200 μg rCsCB or ESP at the first injection, and 100 μg protein (emulsified with incomplete Freund's adjuvant) was given for 2 booster injections at 2-week intervals; antisera were collected before each injection and serum antibody responses to rCsCB and ESP were tested by ELISA.

SDS-PAGE and western blotting

The recombinant protein (1 μg per lane) was subjected to SDS-PAGE and electrotransferred onto polyvinylidene difluoride membrane (PVDF, Whatman; USA) at 100 v for 1 h. The membrane was blocked with 5% skim milk in phosphate buffered saline tween-20 (PBST, pH 7.4) at 37°C for 2 h, washed 5 times with PBST, then incubated with different antisera (antisera against the recombinant CsCB protein, antisera against the ESP, sera from C. sinensis-infected rats and normal rats, respectively, 1:200 dilutions for all sera) at 37°C for 2 h. After washing, the membrane was incubated with rabbit anti-rat IgG HRP-conjugated horse radish peroxidase (1:2,000 dilution; Boster; PR China) at 37°C for 1 h. Diaminobenzidine (DAB) substrate solution (Invitrogen, USA) was used to visualize the reaction according to the manufacturer's instructions.

Immunolocalization of CsCB protein in C. sinensis adult worm

C. sinensis adult worms fixed with 10% neutral formalin were embedded with paraffin and sliced into 3-5 μm thickness. After deparaffinating with dimethyl benzene and gradient alcohol, all sections were blocked with normal goat serum at 4°C overnight, then incubated with rat anti-CsCB serum for 2 h at room temperature (RT), the sera from normal rats were used as negative control, the serum was diluted at 1: 200 for all sections. The sections were subsequently incubated with goat anti-rat IgG labeled with red-fluorescent Cy3 (1: 400 dilutions with 0.1% BSA in PBS, Jackson, USA) for 1 h at RT in dark and imaged using fluorescent microscope (ZEISS; Germany).

Source of serum samples

A total of 276 human serum samples were employed in order to approach the antigenicity and diagnostic value of CsCB in human helminthiases. 80 sera from clonorchiasis patients were collected from endemic areas in Guangxi Province and were egg-positive proved by microscopic examination of stool samples. Other human sera including single infection of E. multilocularis (22), F. hepatica (42), S. japonicum (41), P. westermani (21), T. solium (20) and 50 samples of serum from healthy humans were provided and diagnosed by Centers for Disease Control and Prevention of Nanning, Jiangsu, Gansu and Fujian provinces. Sera were retrieved and stored in aliquots at -20°C until use.

Serodiagnosis of human helminthiases by ELISA and western blotting

The optimal antigen concentration and serum dilution required for ELISA were determined by checker board titration. Briefly, 96-well microtiter plates (Costar, USA) were coated with 100 μl 1 μg/ml, 3 μg/ml, 5 μg/ml, 7 μg/ml rCsCB (in 0.05 mol/l NaHCO3 buffer, pH 9.6) and incubated at 4°C overnight. After three washings with PBST, the microplates were blocked with 5% skimmed milk (w/v, in PBST containing 0.1% BSA) for 2 h at 37°C. Following another washing procedure, the plates were incubated with human helminthiases sera (1:50, 1:100, 1:200, 1:400 dilutions for each coating concentration in PBST containing 0.1% BSA) for 2 h at 37°C. Goat anti-human IgG (1:10,000 dilutions in 0.1% BSA-PBST recommended by producer; Invitrogen, USA) were employed as the secondary antibody. Subsequent reactions were developed with 3', 3', 5', 5'-tetramethyl benzidine (TMB; BD Biosciences; USA) and stopped with 2 M H2SO4. All assays were tested in triplicate and repeated twice. The absorbance value was measured at 450 nm.

In western blotting assay, rCsCB was subjected to 12% SDS-PAGE and electrotransferred onto PVDF membrane (Whatman; USA). The membrane was incubated with different human helminthiases sera (clonorchiasis, echinococciosis, fascioliasis, schistosomiasis, paragonimiasis, and cysticercosis, respectively) which showed high OD450 values by ELISA test at 37°C for 2 h, human healthy serum was used as negative control, all sera were 1:400 diluted in PBST containing 0.1% BSA. Goat anti-human IgG HRP-conjugated horse radish peroxidase (1:2,000 dilution; Boster; PR China) was the secondary antibody to react with the membrane and DAB substrate solution was used to visualize the reaction.

Statistical analysis

Software package SPSS16.0 was used in the present study for all statistical analysis. Briefly, Student's t test was used to analyze the measurement data among groups. P value of < 0.05 was considered statistically significant. The optimal cut-off value was calculated based on ROC curve analysis which correlated with sensitivity and 1-specificity [45]. ROC curve, area under the curve (AUC) and scatter plot were carried out using SPSS16.0. The sensitivity and specificity were calculated using microscopic examination of stool samples as gold standard method.

Ethical approval

Centers for Disease Control and Prevention of Nanning, Jiangsu, Gansu and Fujian provinces granted ethical approval for human sera used in the present study. All animal experiments were approved by institute's ethical committee of Sun Yat-sen University.

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

WJC, XYW and XBY conceived and designed the experiments; WJC, XYW, XRL, CHZ, XLL, CHD, HLL, JTM, YXF and CL performed the experiments; WJC, XYW, XRL and XLL analyzed the data. All authors read and approved the final manuscript.


This work is supported by the National S & T Major Program (Grant No. 2008ZX10004-011) and Development Program of China (973 program; no. 2010CB530000).


  1. Lun ZR, Gasser RB, Lai DH, Li AX, Zhu XQ, Yu XB, Fang YY: Clonorchiasis: a key foodborne zoonosis in China.

    Lancet Infect Dis 2005, 5:31-41. PubMed Abstract | Publisher Full Text OpenURL

  2. Chai JY, Darwin Murrell K, Lymbery AJ: Fish-borne parasitic zoonoses: status and issues.

    Int J Parasitol 2005, 35:1233-1254. PubMed Abstract | Publisher Full Text OpenURL

  3. Sripa B: Pathobiology of opisthorchiasis: an update.

    Acta Trop 2003, 88:209-220. PubMed Abstract | Publisher Full Text OpenURL

  4. Shin HR, Oh JK, Masuyer E, Curado MP, Bouvard V, Fang YY, Wiangnon S, Sripa B, Hong ST: Epidemiology of cholangiocarcinoma: an update focusing on risk factors.

    Cancer Sci 2010, 101:579-585. PubMed Abstract | Publisher Full Text OpenURL

  5. Lee M, Chung YB, Lee SK, Chung BS, Li S, Choi MH, Hong ST: The identification of a Clonorchis sinensis gene encoding an antigenic egg protein.

    Parasitol Res 2005, 95:224-226. PubMed Abstract | Publisher Full Text OpenURL

  6. Zhou XN, Lv S, Yang GJ, Kristensen TK, Bergquist NR, Utzinger J, Malone JB: Spatial epidemiology in zoonotic parasitic diseases: insights gained at the 1st International Symposium on Geospatial Health in Lijiang, China, 2007.

    Parasit Vectors 2009, 2:10. PubMed Abstract | BioMed Central Full Text | PubMed Central Full Text OpenURL

  7. Mesplet M, Echaide I, Dominguez M, Mosqueda JJ, Suarez CE, Schnittger L, Florin-Christensen M: Bovipain-2, the falcipain-2 ortholog, is expressed in intraerythrocytic stages of the tick-transmitted hemoparasite Babesia bovis.

    Parasit Vectors 2010, 3:113. PubMed Abstract | BioMed Central Full Text | PubMed Central Full Text OpenURL

  8. Cruz CE, Fogaca AC, Nakayasu ES, Angeli CB, Belmonte R, Almeida IC, Miranda A, Miranda MT, Tanaka AS, Braz GR, et al.: Characterization of proteinases from the midgut of Rhipicephalus (Boophilus) microplus involved in the generation of antimicrobial peptides.

    Parasit Vectors 2010, 3:63. PubMed Abstract | BioMed Central Full Text | PubMed Central Full Text OpenURL

  9. Sojka D, Franta Z, Horn M, Hajdusek O, Caffrey CR, Mares M, Kopacek P: Profiling of proteolytic enzymes in the gut of the tick Ixodes ricinus reveals an evolutionarily conserved network of aspartic and cysteine peptidases.

    Parasit Vectors 2008, 1:7. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  10. Sajid M, McKerrow JH: Cysteine proteases of parasitic organisms.

    Mol Biochem Parasitol 2002, 120:1-21. PubMed Abstract | Publisher Full Text OpenURL

  11. Dalton JP, Neill SO, Stack C, Collins P, Walshe A, Sekiya M, Doyle S, Mulcahy G, Hoyle D, Khaznadji E, et al.: Fasciola hepatica cathepsin L-like proteases: biology, function, and potential in the development of first generation liver fluke vaccines.

    Int J Parasitol 2003, 33:1173-1181. PubMed Abstract | Publisher Full Text OpenURL

  12. Meemon K, Grams R, Vichasri-Grams S, Hofmann A, Korge G, Viyanant V, Upatham ES, Habe S, Sobhon P: Molecular cloning and analysis of stage and tissue-specific expression of cathepsin B encoding genes from Fasciola gigantica.

    Mol Biochem Parasitol 2004, 136:1-10. PubMed Abstract | Publisher Full Text OpenURL

  13. Na BK, Kim SH, Lee EG, Kim TS, Bae YA, Kang I, Yu JR, Sohn WM, Cho SY, Kong Y: Critical roles for excretory-secretory cysteine proteases during tissue invasion of Paragonimus westermani newly excysted metacercariae.

    Cell Microbiol 2006, 8:1034-1046. PubMed Abstract | Publisher Full Text OpenURL

  14. Loukas A, Bethony JM, Williamson AL, Goud GN, Mendez S, Zhan B, Hawdon JM, Elena Bottazzi M, Brindley PJ, Hotez PJ: Vaccination of dogs with a recombinant cysteine protease from the intestine of canine hookworms diminishes the fecundity and growth of worms.

    J Infect Dis 2004, 189:1952-1961. PubMed Abstract | Publisher Full Text OpenURL

  15. Abdulla MH, Lim KC, Sajid M, McKerrow JH, Caffrey CR: Schistosomiasis mansoni: novel chemotherapy using a cysteine protease inhibitor.

    PLoS Med 2007, 4:e14. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  16. Behnke JM, Buttle DJ, Stepek G, Lowe A, Duce IR: Developing novel anthelmintics from plant cysteine proteinases.

    Parasit Vectors 2008, 1:29. PubMed Abstract | BioMed Central Full Text | PubMed Central Full Text OpenURL

  17. Kang JM, Bahk YY, Cho PY, Hong SJ, Kim TS, Sohn WM, Na BK: A family of cathepsin F cysteine proteases of Clonorchis sinensis is the major secreted proteins that are expressed in the intestine of the parasite.

    Mol Biochem Parasitol 2010, 170:7-16. PubMed Abstract | Publisher Full Text OpenURL

  18. Ju JW, Joo HN, Lee MR, Cho SH, Cheun HI, Kim JY, Lee YH, Lee KJ, Sohn WM, Kim DM, et al.: Identification of a serodiagnostic antigen, legumain, by immunoproteomic analysis of excretory-secretory products of Clonorchis sinensis adult worms.

    Proteomics 2009, 9:3066-3078. PubMed Abstract | Publisher Full Text OpenURL

  19. Li Y, Hu X, Liu X, Xu J, Hu F, Ma C, Yu X: Molecular cloning and analysis of stage and tissue-specific expression of Cathepsin L-like protease from Clonorchis sinensis.

    Parasitol Res 2009, 105:447-452. PubMed Abstract | Publisher Full Text OpenURL

  20. Lee JS, Kim IS, Sohn WM, Lee J, Yong TS: Vaccination with DNA encoding cysteine proteinase confers protective immune response to rats infected with Clonorchis sinensis.

    Vaccine 2006, 24:2358-2366. PubMed Abstract | Publisher Full Text OpenURL

  21. Li S, Chung YB, Chung BS, Choi MH, Yu JR, Hong ST: The involvement of the cysteine proteases of Clonorchis sinensis metacercariae in excystment.

    Parasitol Res 2004, 93:36-40. PubMed Abstract | Publisher Full Text OpenURL

  22. Kang TH, Yun DH, Lee EH, Chung YB, Bae YA, Chung JY, Kang I, Kim J, Cho SY, Kong Y: A cathepsin F of adult Clonorchis sinensis and its phylogenetic conservation in trematodes.

    Parasitology 2004, 128:195-207. PubMed Abstract | Publisher Full Text OpenURL

  23. Morphew RM, Wright HA, LaCourse EJ, Woods DJ, Brophy PM: Comparative proteomics of excretory-secretory proteins released by the liver fluke Fasciola hepatica in sheep host bile and during in vitro culture ex host.

    Mol Cell Proteomics 2007, 6:963-972. PubMed Abstract | Publisher Full Text OpenURL

  24. Nagano I, Pei F, Wu Z, Wu J, Cui H, Boonmars T, Takahashi Y: Molecular expression of a cysteine proteinase of Clonorchis sinensis and its application to an enzyme-linked immunosorbent assay for immunodiagnosis of clonorchiasis.

    Clin Diagn Lab Immunol 2004, 11:411-416. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  25. Na BK, Lee HJ, Cho SH, Lee HW, Cho JH, Kho WG, Lee JS, Song KJ, Park PH, Song CY, Kim TS: Expression of cysteine proteinase of Clonorchis sinensis and its use in serodiagnosis of clonorchiasis.

    J Parasitol 2002, 88:1000-1006. PubMed Abstract | Publisher Full Text OpenURL

  26. Cornelissen JB, Gaasenbeek CP, Borgsteede FH, Holland WG, Harmsen MM, Boersma WJ: Early immunodiagnosis of fasciolosis in ruminants using recombinant Fasciola hepatica cathepsin L-like protease.

    Int J Parasitol 2001, 31:728-737. PubMed Abstract | Publisher Full Text OpenURL

  27. Carnevale S, Rodriguez MI, Guarnera EA, Carmona C, Tanos T, Angel SO: Immunodiagnosis of fasciolosis using recombinant procathepsin L cystein proteinase.

    Diagn Microbiol Infect Dis 2001, 41:43-49. PubMed Abstract | Publisher Full Text OpenURL

  28. Sripa J, Brindley PJ, Sripa B, Loukas A, Kaewkes S, Laha T: Evaluation of liver fluke recombinant cathepsin B-1 protease as a serodiagnostic antigen for human opisthorchiasis.

    Parasitol Int 2011. OpenURL

  29. McKerrow JH, Caffrey C, Kelly B, Loke P, Sajid M: Proteases in parasitic diseases.

    Annu Rev Pathol 2006, 1:497-536. PubMed Abstract | Publisher Full Text OpenURL

  30. Illy C, Quraishi O, Wang J, Purisima E, Vernet T, Mort JS: Role of the occluding loop in cathepsin B activity.

    J Biol Chem 1997, 272:1197-1202. PubMed Abstract | Publisher Full Text OpenURL

  31. Baig S, Damian RT, Peterson DS: A novel cathepsin B active site motif is shared by helminth bloodfeeders.

    Exp Parasitol 2002, 101:83-89. PubMed Abstract | Publisher Full Text OpenURL

  32. Ranjit N, Zhan B, Stenzel DJ, Mulvenna J, Fujiwara R, Hotez PJ, Loukas A: A family of cathepsin B cysteine proteases expressed in the gut of the human hookworm, Necator americanus.

    Mol Biochem Parasitol 2008, 160:90-99. PubMed Abstract | Publisher Full Text OpenURL

  33. Beckham SA, Law RH, Smooker PM, Quinsey NS, Caffrey CR, McKerrow JH, Pike RN, Spithill TW: Production and processing of a recombinant Fasciola hepatica cathepsin B-like enzyme (FhcatB1) reveals potential processing mechanisms in the parasite.

    Biol Chem 2006, 387:1053-1061. PubMed Abstract | Publisher Full Text OpenURL

  34. McGonigle L, Mousley A, Marks NJ, Brennan GP, Dalton JP, Spithill TW, Day TA, Maule AG: The silencing of cysteine proteases in Fasciola hepatica newly excysted juveniles using RNA interference reduces gut penetration.

    Int J Parasitol 2008, 38:149-155. PubMed Abstract | Publisher Full Text OpenURL

  35. Correnti JM, Brindley PJ, Pearce EJ: Long-term suppression of cathepsin B levels by RNA interference retards schistosome growth.

    Mol Biochem Parasitol 2005, 143:209-215. PubMed Abstract | Publisher Full Text OpenURL

  36. Skelly PJ, Da'dara A, Harn DA: Suppression of cathepsin B expression in Schistosoma mansoni by RNA interference.

    Int J Parasitol 2003, 33:363-369. PubMed Abstract | Publisher Full Text OpenURL

  37. Pak JH, Kim DW, Moon JH, Nam JH, Kim JH, Ju JW, Kim TS, Seo SB: Differential gene expression profiling in human cholangiocarcinoma cells treated with Clonorchis sinensis excretory-secretory products.

    Parasitol Res 2009, 104:1035-1046. PubMed Abstract | Publisher Full Text OpenURL

  38. Kim YJ, Choi MH, Hong ST, Bae YM: Resistance of cholangiocarcinoma cells to parthenolide-induced apoptosis by the excretory-secretory products of Clonorchis sinensis.

    Parasitol Res 2009, 104:1011-1016. PubMed Abstract | Publisher Full Text OpenURL

  39. Hu F, Hu X, Ma C, Zhao J, Xu J, Yu X: Molecular characterization of a novel Clonorchis sinensis secretory phospholipase A(2) and investigation of its potential contribution to hepatic fibrosis.

    Mol Biochem Parasitol 2009, 167:127-134. PubMed Abstract | Publisher Full Text OpenURL

  40. Ma C, Hu X, Hu F, Li Y, Chen X, Zhou Z, Lu F, Xu J, Wu Z, Yu X: Molecular characterization and serodiagnosis analysis of a novel lysophospholipase from Clonorchis sinensis.

    Parasitol Res 2007, 101:419-425. PubMed Abstract | Publisher Full Text OpenURL

  41. Smooker PM, Jayaraj R, Pike RN, Spithill TW: Cathepsin B proteases of flukes: the key to facilitating parasite control?

    Trends Parasitol 2010, 26:506-514. PubMed Abstract | Publisher Full Text OpenURL

  42. Wang X, Liang C, Chen W, Fan Y, Hu X, Xu J, Yu X: Experimental model in rats for study on transmission dynamics and evaluation of Clonorchis sinensis infection immunologically, morphologically, and pathologically.

    Parasitol Res 2009, 106:15-21. PubMed Abstract | Publisher Full Text OpenURL

  43. Sripa B, Kaewkes S, Sithithaworn P, Mairiang E, Laha T, Smout M, Pairojkul C, Bhudhisawasdi V, Tesana S, Thinkamrop B, et al.: Liver fluke induces cholangiocarcinoma.

    PLoS Med 2007, 4:e201. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  44. Olnes MJ, Erlich R: A review and update on cholangiocarcinoma.

    Oncology 2004, 66:167-179. PubMed Abstract | Publisher Full Text OpenURL

  45. Bon B, Houze S, Talabani H, Magne D, Belkadi G, Develoux M, Senghor Y, Chandenier J, Ancelle T, Hennequin C: Evaluation of a rapid enzyme-linked immunosorbent assay for diagnosis of strongyloidiasis.

    J Clin Microbiol 2010, 48:1716-1719. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL