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

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.     

华支睾吸虫LAP2基因的生物信息学分析及克隆表达、组织定位

目的利用生物信息学方法分析华支睾吸虫LAP2全长基因的结构和功能,并将该基因克隆至原核表达载体进行表达、纯化,为进一步研究LAP2功能奠定基础。方法利用生物信息学相关软件,分析华支睾吸虫LAP2基因及其蛋白的结构、生物学和免疫学功能特征。针对LAP2的EST序列的编码区设计引物,从华支睾吸虫囊蚴cDNA质粒中扩增目的基因,克隆到原核表达质粒pET-28a（＋）中,经PCR、双酶切及DNA测序鉴定的阳性克隆诱导目的蛋白表达、亲和层析纯化、免疫印迹鉴定及组化定位。结果华支睾吸虫LAP2基因全长为1761bp,其编码序列长度为1662bp,编码553个氨基酸,理论分子量是59696.5Da,与人的该基因氨基酸序列相似性为26.7%,一致性仅为15.4%。该基因在大肠杆菌中可被高效表达,纯化的重组蛋白能被感染华支睾吸虫的大鼠血清识别,免疫组化结果表明该重组蛋白定位于华支睾吸虫成虫的肠支及表膜。结论华支睾吸虫LAP2在大肠杆菌中呈高效的可溶性表达,具有良好的抗原活性,可能为华支睾吸虫成虫分泌排泄抗原的组份之一。     

Molecular identification, immunolocalization, and characterization of Clonorchis sinensis calmodulin

One cDNA clone (Cs18h09) encoding Clonorchis sinensis calmodulin (CsCaM) was isolated from our adult cDNA plasmid library. The open reading frame of CsCaM contains 450 bp which encodes 149 amino acids. CsCaM protein comprises four calcium-binding EF-hand motifs. The amino acid sequence of CsCaM shares very high homology with other species. Quantitative RT-polymerase chain reaction (PCR) revealed that CsCaM mRNA was constitutively transcribed in development cycle stages of the parasite, including adult worm, metacercaria, excysted metacercaria, and egg. In addition, recombinant CsCaM (rCsCaM) was expressed as a soluble protein and anti-rCsCaM rat serum could detect CsCaM in the C. sinensis somatic extracts but not in the C. sinensis excretory–secretory products (ESPs). Moreover, immunolocalization assay showed that CsCaM was located in tegument, intestine, pharynx, and eggs. Furthermore, rCsCaM was found to bind calcium ion (Ca²⁺) and magnesium (Mg²⁺) in electrophoretic mobility shift assay. Ca²⁺ binding increased the ability of rCsCaM to bind the hydrophobic fluorescent probe 8-anilinonaphthalene-1-sulphonate, causing a blue shift in the fluorescence emission from 540 to 515 nm with an excitation wavelength of 380 nm and substantial increase in fluorescence intensity but not Mg²⁺. Collectively, here we showed the basic characterization of CsCaM and inferred that CsCaM could be a Ca²⁺ sensor protein, and CsCaM may possibly participate in growth and development of adult worm and egg of C. sinensis through binding Ca²⁺.     

Molecular cloning, expression, and immunolocalization of the NAD(+)-dependent glycerol 3-phosphate dehydrogenase (GPD) from Clonorchis sinensis

Glycerol 3-phosphate dehydrogenase (GPD) plays an important role in the energy metabolism and nutrition metabolism. In order to know about the biological functions of GPD of Clonorchis sinensis (C. sinensis), we identified a complete gene coding GPD from C. sinensis metacercaria cDNA library. This novel cDNA sequence contains 1,056 bp with a putative open reading frame of 351 amino acids and shares 74% identity with GPD from Schistosoma mansoni. Recombinant CsGPD was expressed and purified from Escherichia coli BL21 (DE3). Western blot analysis displayed that recombinant CsGPD can be recognized by anti-CsGPD serum and C. sinensis-infected serum. RT-PCR and immunolocalization analysis confirmed that GPD expressed both at the stage of adult worm and metacercaria of C. sinensis and immunolocated at the tegument of adult worm, tegument and tegumentary cells of metacercaria. Our current study has paved the way for the further researches about the biological functions involved in the growth of C. sinensis.     

Molecular expression of a cysteine proteinase of Clonorchis sinensis and its application to an enzyme-linked immunosorbent assay for immunodiagnosis of clonorchiasis

We produced a recombinant cysteine proteinase of Clonorchis sinensis and tested its value as an antigen for serologic diagnosis of C. sinensis infections. The predicted amino acid sequence of the cysteine proteinase of C. sinensis was 58, 48, and 40% identical to those of cathepsin L cysteine proteinases from Paragonimus westermani, Schistosoma japonicum, and Fasciola hepatica, respectively. Western blotting analysis showed that sera from patients infected with C. sinensis strongly reacted with the recombinant protein and that sera from patients infected with S. japonicum weakly reacted with the recombinant protein. Antibody against the recombinant protein stained proteins migrating at about 37 and 28 kDa in C. sinensis adult worm crude extracts. Immunostaining revealed that the cysteine proteinase of C. sinensis was located in the intestinal epithelial cells of the adult parasite and in intrauterine eggs. The specificity and sensitivity of the recombinant antigen or C. sinensis adult worm crude extracts were assessed by an enzyme-linked immunosorbent assay (ELISA) using serum samples from humans infected with different parasites, including 50 patients with clonorchiasis, and negative controls. The sensitivities of the ELISA with the recombinant antigen and C. sinensis adult worm crude extracts were 96 and 88%, respectively. The specificities of the ELISA with the recombinant antigen and C. sinensis adult worm crude extracts were 96.2 and 100%, respectively. The results suggested that the recombinant cysteine proteinase-based ELISA could provide a highly sensitive and specific assay for diagnosis of clonorchiasis.     

Identification,sequence analysis,and characterization of serine/threonine protein kinase 17A from Clonorchis sinensis

This is the first report of a novel protein from Clonorchis sinensis (C. sinensis), serine/threonine protein kinase 17A (CsSTK17A), which belongs to a member of the death-associated protein kinase (DAPK) family known to regulate diverse biological processes. The full-length sequence encoding CsSTK17A was isolated from C. sinensis adult cDNA plasmid library. Two transcribed isoforms of the gene were identified from the genome of C. sinensis. CsSTK17A contains a kinase domain at the N-terminus that shares a degree of conservation with the DAPK families. Besides, the catalytic domain contains 11 subdomains conserved among STKs and shares the highest identity with STK from Schistosoma mansoni (55.9 %). Three-dimensional structure of CsSTK17A displays the canonical STK fold, including the helix C, P-loop, and the activation loop. We obtained recombinant CsSTK17A (rCsSTK17A) and anti-rCsSTK17A IgG. The rCsSTK17A could be probed by anti-rCsSTK17A rat serum, C. sinensis-infected rat serum and the sera from rats immunized with C. sinensis excretory-secretory products, indicating that it is a circulating antigen possessing a strong immunocompetence. Moreover, quantitative RT-PCR and western blotting analyses revealed that CsSTK17A exhibited the highest mRNA and protein expression level in eggs, followed by metacercariae and adult worms. Intriguingly, in the immunolocalization assay, CsSTK17A was intensively localized to the operculum region of eggs in uterus, as well as the vitelline gland of both adult worm and metacercaria, implying that the protein was associated with the reproduction and development of C. sinensis. Overall, these fundamental studies might contribute to further researches on signaling systems of the parasite.     

41.5-kDa Cathepsin L protease from Clonorchis sinensis: expression, characterization, and serological reactivity of one excretory–secretory antigen

Cysteine proteases (CPs) were associated with the pathogenicity and excystment of Clonorchis sinensis. Most of them were potential antigens for the immunodiagnosis of clonorchiasis. More researches on CPs will let us know more about their functions, and further employ them for the development of more efficient diagnostic reagent and prevention strategies. In the current study, a full-length sequence encoding cathepsin L from C. sinensis (CsCL41.5) was identified from our adult cDNA library. Bioinformatic analysis showed that CsCL41.5 included typical motifs of cathepsin L (ERFNIN and GNFD motifs) and conserved amino acid positions which constituted the active center of the enzyme. The identity of its amino acid sequence with the cathepsin L of Schistosoma japonicum was 49.6 %. Recombinant CsCL41.5 (rCsCL41.5) was highly expressed in the form of inclusion body in Escherichia coli, and soluble rCsCL41.5 was obtained after purification and renaturation. Western blotting analysis indicated that CsCL41.5 is an excretory-secretory antigen of C. sinensis adult. Immunolocalization demonstrated that CsCL41.5 is distributed in the intestine and eggs in the uterus of adult worm, tegument of metacercaria, oral suck, and tail of cercaria. ELISA assays showed that IgG4 was the predominant IgG isotype responding to rCsCL41.5 in sera from clonorchiasis patients. The sensitivity and specificity of specific IgG4 detection with rCsCL41.5 was 62.5 % (15/24) and 81.7 % (49/60), respectively. It was concluded that there were differences in biological function, efficiency of serodiagnosis, and characterization of immune reactivity between CsCL41.5 and other CPs of C. sinensis, combining with previous studies.     

Allergens of Schistosoma mansoni. II. Fractionation and characterization of S. mansoni egg allergens

The interaction of Schistosoma mansoni crude soluble egg antigen (SEA) with IgE antibodies in sera from S. mansoni-infected mice, rats and humans has been studied by the radioallergosorbent test (RAST) and the Prausnitz-Küstner (PK) technique. IgE antibodies recognizing egg antigens were present as early as day 21 after the infection in the mouse sera and day 28 in rat sera. IgE in sera of infected humans reacted with antigenic components in the Mr range 70,000-150,000 and focusing as a broad peak in the pH range 4.5-6.5 as measured by RAST. SDS-PAGE followed by western blotting showed the presence of major components at molecular weights of 117,000 and 35,000-43,000. In the PK test, using mouse sera, components focusing in the alkaline pH range also gave a positive reaction. Most of the allergenic activity was bound by concanavalin A-Sepharose and by wheat germ agglutinin-Ultrogel. IgE in serum from an infected non-permissive host (the Fischer rat) apparently recognized egg-stage-specific allergen as indicated by differences in the time course of the IgE response to egg allergens compared to the adult material. When analyzed by SDS-PAGE and western blotting with day 45-infected rat serum, SEA showed some qualitative and quantitative differences to adult worm antigen. Molecules at molecular weights between 25,000 and 30,000 and at about 43,000 in SEA reacted with rat serum IgE and were absent from adult worm antigen. The allergenic similarities between egg and adult worm are discussed.     

华支睾吸虫膜抗原/排泄分泌抗原酸性磷酸酶的克隆、表达、生物学特征分析及组织定位

 目的:对华支睾吸虫(Clonorchis sinensis, Cs)成虫酸性磷酸酶 (acid phosphatase, AP)进行克隆、表达、生物学特征分析、组织定位及膜抗原/排泄分泌抗原鉴定。方法:对CsAP进行生物信息学、分子生物学、免疫组化及明胶酶谱分析。结果:从Cs cDNA文库中筛选出编码AP新基因,全长1 410 bp,重组并由大肠杆菌表达、纯化,得到分子量为55 kD的重组蛋白CsAP。Western blotting分析表明,CsAP既是膜抗原又是分泌排泄抗原;免疫组化显示,CsAP荧光显示于成虫的表皮层和肠支,在囊蚴也有显示,在雷蚴和尾蚴未显示荧光;ELISA分析表明CsAP识别华支睾吸虫病人和日本血吸虫病人存在吸虫间的交叉免疫反应,CsAP及粗抗原识别轻、中、重度感染程度华支睾吸虫病人的差别不明显。重组蛋白免疫大鼠后,总IgG抗体滴度于3周达较高峰,抗体效价大于1∶25 600。明胶降解实验表明：CsAP具降解胶原能力。结论: 上述结果表明,CsAP在大肠杆菌中高效表达,具有较好的免疫原性,但血清诊断价值不理想;CsAP可能既是膜抗原,又是排泄分泌抗原。     

Cloning, characterization, and expression of a novel secretory lipase-like gene from Clonorchis sinensis

A secretory lipase-like gene was isolated from total cDNA of adult Clonorchis sinensis. The gene has an open reading frame of 1,218 bp long and encodes for a protein of 406 amino acids including a putative signal peptide of 20 amino acids. The deduced amino acid sequence including signal peptide has 42–45% identity with lipase of other species and two typical enzymic active sites that contain consensus sequence (Gly-X-Ser-X-Gly) of lipase. The cDNA encoding this protein was subcloned into pET-28a (+) expression vector and expressed in Escherichia coli. The expressed fusion protein has a molecular mass of about 45 kDa. Prediction of signal peptide and Western blot analysis indicated that the secretory lipase-like protein is an excretory–secretory product of C. sinensis. Immunostaining revealed that the secretory lipase-like protein was localized in the tegument of the adult worm and metacercaria. These results provide basis for further studies on the nutrition taking and invasion of C. sinensis mediated by the secretory lipase-like protein.     

中华唐样吸虫（Tangiopsis chinensis（Tang，1951））（Trematoda：Hemiuridae）后期生活史研究

本文报告福建璋平中华唐样吸虫（Ｔａｎｇｉｏｐｓｉｓｃｈｉｎｅｎｓｉｓ（Ｔａｎｇ，１９５１））在第二中间宿主的发育，第二中间宿主为介虫Ｃｙｐｒｅｔｔａｓｐ．，Ｈｅｔｅｒｏｃｙｐｒｉｓｓｐ．，Ｄｏｌｅｒｏｃｙｐｒｉｓｓｉｎｅｎｓｉｓ，并对该吸虫后阶段发育进行观察描述。     

Identification and functional characterization of CsStefin-1, a cysteine protease inhibitor of Clonorchis sinensis

Cathepsin Fs of Clonorchis sinensis (CsCFs) are major secreted proteins that are expressed in the intestine of the parasite and play pivotal roles in parasite nutrition and host-parasite interactions. However, strict regulation of their activities is also essential to minimize inadequate superfluous damage to the parasite and host. In this study, we identified and characterized a novel cysteine protease inhibitor of C. sinensis, CsStefin-1, as a modulator of CsCFs. CsStefin-1 was shown to be a typical cysteine protease inhibitor of family 1 cystatins that lacks the N-terminal signal peptide and C-terminal cysteine residues required for disulfide bond formation. Phylogenetic and structural analyses also showed that CsStefin-1 is a family 1 intracellular cystatin. Bacterially expressed CsStefin-1 effectively inhibited various cysteine proteases, including human cathepsin B, human cathepsin L, papain, and CsCFs. CsStefin-1 was active over a wide pH range and was highly stable under physiological conditions. CsStefin-1 also inhibited the processing of CsCFs. CsStefin-1 was expressed throughout various developmental stages of the parasite from metacercaria to adult worm and the protein was detected in worm extract, but not in the excretory and secretory products of adult worm. Immunolocalization analysis showed that CsStefin-1 was mainly localized to the intestinal epithelium, where CsCFs are actively synthesized. Our results collectively suggest the regulatory functions of CsStefin-1, modulation of CsCFs activity and processing, to protect the parasite from superfluous damage by the endogenous cysteine proteases.