title: 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.
authors.affiliation: Department of Parasitology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong, 510080, China.
authors.forename: Xiaoli
authors.initials: X
authors.lastname: Lv
authors.forename: Wenjun
authors.initials: W
authors.lastname: Chen
authors.forename: Xiaoyun
authors.initials: X
authors.lastname: Wang
authors.forename: Xuerong
authors.initials: X
authors.lastname: Li
authors.forename: Jiufeng
authors.initials: J
authors.lastname: Sun
authors.forename: Chuanhuan
authors.initials: C
authors.lastname: Deng
authors.forename: Jingtao
authors.initials: J
authors.lastname: Men
authors.forename: Yanli
authors.initials: Y
authors.lastname: Tian
authors.forename: Chenhui
authors.initials: C
authors.lastname: Zhou
authors.forename: Huali
authors.initials: H
authors.lastname: Lei
authors.forename: Chi
authors.initials: C
authors.lastname: Liang
authors.forename: Xinbing
authors.initials: X
authors.lastname: Yu
mesh_terms: D000818:Animals; D000909:Antibodies, Helminth / Q000097:blood*; D000947:Antigens, Helminth / Q000235:genetics / Q000302:isolation & purification; D015153:Blotting, Western; D003001:Cloning, Molecular; D003003:Clonorchiasis / Q000175:diagnosis*; D003004:Clonorchis sinensis / Q000201:enzymology* / Q000235:genetics / Q000276:immunology; D057056:Cysteine Proteases / Q000235:genetics / Q000302:isolation & purification; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D015870:Gene Expression; D020869:Gene Expression Profiling; D006801:Humans; D007074:Immunoglobulin G / Q000097:blood; D011817:Rabbits; D051381:Rats; D017207:Rats, Sprague-Dawley; D060888:Real-Time Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification; D012680:Sensitivity and Specificity; D017386:Sequence Homology, Amino Acid; D012698:Serologic Tests / Q000379:methods
chemical_list: D000909:Antibodies, Helminth; D000947:Antigens, Helminth; D007074:Immunoglobulin G; D011994:Recombinant Proteins; D057056:Cysteine Proteases