一种华支睾吸虫亲肌素样基因的克隆、表达与免疫学功能初步研究

目的对新发现的华支睾吸虫亲肌素样蛋白（CsMLP）进行克隆、原核表达,初步了解其重组产物的免疫学功能。方法应用生物信息学分析软件,分析CsMLP的序列特点和基本理化特征;将CsMLP基因克隆到原核表达质粒pET．28a（＋）中,诱导表达并用镍离子金属螯合剂亲和层析柱进行纯化,用纯化的CsMLP蛋白免疫SD大鼠获得抗血清;用Westernblot进行免疫反应性及免疫原性分析;应用免疫荧光方法观察CsMLP在华支睾吸虫成虫的定位。结果该cDNA序列全长为900bp,编码190个氨基酸,具有Calponin功能域;PCR、双酶切及DNA测序结果均表明pET．28a（＋）-CsMLP重组质粒构建成功;SDS—PAGE结果表明目的基因在大肠杆菌BL21／DE3中获得高效表达,分子量为21300Mr;经亲和层析获得了高纯度蛋白;重组蛋白可被其免疫的SD大鼠血清、感染了华支睾吸虫的SD大鼠血清识别;CsMLP主要定位于虫体富含肌肉组织的口、腹吸盘、咽部,在表膜、肠壁也有分布。结论CsMLP可在原核表达系统中呈现高效的可溶性表达,具有免疫原性．其主要分布在华支睾吸虫肌肉丰富的组织。     

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.     

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.     

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.     

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 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 cloning and immunological characterization of phosphoglycerate kinase from Clonorchis sinensis

The parasite Clonorchis sinensis was determined to utilize a large amount of external glucose to carry its energy metabolism. Phosphoglycerate kinase (PGK), a glycolytic enzyme, found in many parasites, has been identified as one of the candidate molecules distinguished from human counterparts for vaccine and drug developments. A cDNA clone purified by screening a C. sinensis cDNA library using a heterologous cDNA probe encoded a putative peptide of 415 amino acids with over 60% identities with PGKs from a number of animals. The putative peptides revealed domains corresponding to 12 beta-sheets and inner loops forming a substrate-binding cleft of animal PGKs. The gene product was overexpressed in Escherichia coli and showed a PGK-like enzyme activity. A polyclonal antibody raised against the recombinant C. sinensis PGK was specific to native C. sinensis PGK and localized it to the muscular tissue and tegument of the adult flukes. The C. sinensis PGK elicited antibodies in C. sinensis-infected rabbits. Therefore, it is proposed that C. sinensis PGK could be used as an immunoreagent in the serodiagnosis for clonorchiasis.     

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。     

Expression, immunolocalization, and serological reactivity of a novel sphingomyelin phosphodiesterase-like protein, an excretory/secretory antigen from Clonorchis sinensis

Clonorchiasis, caused by Clonorchis sinensis infection, is a zoonotic parasitic disease of hepatobiliary system in which the proteins released by adult are major pathogenetic factors. In this study, we first characterized a putative sphingomyelin phosphodiesterase (CsSMPase) A-like secretory protein, which was highly expressed in the adult worm. The full-length gene was cloned. The putative protein is of relatively low homology comparing with SMPase from other species, and of rich T cell and B cell epitopes, suggesting that it is an antigen of strong antigenicity. The complete coding sequence of the gene was expressed in the Escherichia coli. The recombinant CsSMPase (rCsSMPase) can be recognized by C. sinensis-infected serum, and the protein immunoserum can recognize a specific band in excretory/secretory products (ESPs) of C. sinensis adult by western blotting. Immunolocalization revealed that CsSMPase was not only localized on tegument, ventral sucker of metacercaria, and the intestine of adult but also on the nearby epithelium of bile duct of the infected Sprague–Dawley rats, implying that CsSMPase was mainly secreted and excreted through adult intestine and directly interacted with bile duct epithelium. Although immunized rats evoked high level antibody response, the antigen level was low in clonorchiasis patients. And the sensitivity and specificity of rCsSMPase were 50.0 % (12/24) and 88.4 % (61/69), in sera IgG-ELISA, respectively. It is likely due to the fact that CsSMPase binding to the plasma membrane of biliary epithelium decreases the antigen immune stimulation.     

Recognition and characterization of TGF-β receptor interacting protein 1 (TRIP-1) containing WD40 repeats from <Emphasis Type="Italic">Clonorchis sinensis</Emphasis> by bioinformatics,cloning, and expression in <Emphasis Type="Italic">Escherichia coli</Emphasis>

A cDNA clone encoding a homologue of transforming growth factor beta (TGF-beta) receptor interacting protein 1 (TRIP-1) was recognized and isolated from full-length cDNA plasmid library of Clonorchis sinensis adult. TRIP-1 is a bifunctional molecule in all eukaryote, which modulates the signaling pathway of TGF-beta as a phosphorylation substrate of TGF-beta type II receptor kinase and controls ribosome assembly and mRNA translation as p36 subunit of the eukaryotic translation initiation factor 3. The structural and immunological characteristics of TRIP-1 from C. sinensis (CsTRIP-1) were analyzed by bioinformatics. The complete coding sequence was expressed in Escherichia coli, and the purified recombinant product was obtained. Western blotting with mixed sera from clonorchiasis patients was positive, whereas the normal was negative, suggesting it is a candidate of diagnostic antigen for clonorchiasis. CsTRIP-1 will aid to explore interaction between host and the parasite as well as the mechanism by which TGF-beta controls the development of C. sinensis and participates in the pathogenesis.     

Multiple recombinant antigens of Clonorchis sinensis for serodiagnosis of human clonorchiasis

Antigenic proteins from Clonorchis sinensis have been previously purified and evaluated for their antigenicity to enable the serodiagnosis of clonorchiasis. Though they were of high specificity, molecularly defined proteins were reported to be less sensitive as single antigens than crude antigen. To resolve this issue, 11 clones were selected by immunoscreening an adult C. sinensis cDNA library using infected human sera. Mixed antigens were prepared using recombinant proteins of positive clones and investigated for antigenicity by immunoblotting against C. sinensis- and helminth-infected patient sera. A mixed antigen of recombinant 28 and 26 kDa glutathion S-transferases (Cs28GST and Cs26GST) produced 76% sensitivity and 95% specificity. Furthermore, a triple mix of recombinant Cs26GST and Cs28GST with vitelline precursor protein pushed up the sensitivity to 87% and maintained specificity at 95%. It is proposed that multiple antigen mixes should be further studied to develop rapid serodiagnostic test kits for the serodiagnosis of human clonorchiasis.     

The identification of antigenic proteins: 14-3-3 protein and propionyl-CoA carboxylase in Clonorchis sinensis

Clonorchis sinensis, the causative agent of clonorchiasis, is widespread in East and Southeast Asia, including China, Vietnam and the Republic of Korea. We identified antigenic proteins from adult C. sinensis liver flukes using immunoproteomic analysis. In this study, we found 23 candidate antigenic proteins with a pI in the range of 5.4-6.2 in total lysates of C. sinensis. The antigenic protein spots reacted against sera from clonorchiasis patients and were identified as cysteine proteases, glutathione transferases, gelsolin, propionyl-CoA carboxylase (PCC), prohibitin and 14-3-3 protein (14-3-3) using LC-coupled ESI-MS/MS and an EST database for C. sinensis. PCC and 14-3-3 were identified for the first time as serological antigens for the diagnosis of C. sinensis. To validate the antigenicity of PCC and 14-3-3, recombinant proteins were immunoblotted with sera from clonorchiasis patients. The structural, functional and immunological characteristics of the putative amino acid sequence were predicted by bioinformatics analysis. Our novel finding will contribute to the development of diagnostics for clonorchiasis. These results suggest that immunoproteomic approaches are valuable tools to identify antigens that could be used as targets for effective parasitic infection control strategies.     

华支睾吸虫卵黄前体蛋白B基因的识别、序列分析和克隆表达

为识别和克隆华支睾吸虫新基因 ,对华支睾cDNA质粒文库进行随机筛选并测序 ,并利用在线生物信息学工具进行序列分析 ,识别华支睾吸虫未知基因 ,同时根据PGEX -4T- 1多克隆位点及未知基因的序列设计引物 ,PCR扩增目的基因 ,并构建原核重组质粒。结果发现了CsvpB基因 ,其完整阅读框含 76 2个碱基 ,编码 2 5 4个氨基酸 ,理论分子量为 2 7. 7kDa。序列分析表明 ,CsvpB蛋白与其它物种的卵黄前体蛋白有较高的同源性 ,所构建的重组原核表达质粒PGEX- 4T- 1 vpB经PCR、双酶切及测序证实与目标基因相符。     

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.     

Molecular cloning, expression, and characterization of cyclophilin A from Clonorchis sinensis

This study described the recognization, cloning, and recombinant expression of cyclophilin A-like gene from Clonorchis sinensis adult complementary DNA library (CsCyPA) and its expression and secretion in adult. Western blotting demonstrated the recombinant CsCyPA could be recognized by sera of clonorchiasis patients and a sole protein of the same size in the excretory-secretory antigens of in vitro cultured adult could be recognized by antiserum raised against the recombinant CsCyPA. Immunohistochemistry demonstrated that the CsCyPA was secreted in scattered vesicles from subtegumental parenchyma cells to the surface of tegument and mainly released from the tegument. ELISA showed the serum levels of IgG against CsCyPA in clonorchiasis patients negatively correlated with worm loads. This study suggested that C. sinensis adult in biliary ducts could release CsCyPA without signal peptide through nonclassical secretory pathway into the liver and might play a role in inflammation and biliary epithelium proliferation and adenomatoid hyperplasia.