Comparison of the cloned genes of the 26- and 28-kilodalton glutathione S-transferases of Schistosoma japonicum and Schistosoma mansoni.

Both Schistosoma japonicum and S. mansoni contain 28- and 26-kDa glutathione S-transferases (GSTs). Despite their immunological cross-reactivity using rabbit antisera, the S. japonicum 28-kDa GST (Sj28) is weakly immunogenic relative to the S. mansoni protein (Sm28) in mouse immunization experiments using GSTs purified from adult worms. The difference in immunogenicity is also observed during schistosome infection in mice. Using surface-labeled living S. japonicum worms, evidence was obtained for a surface location of Sj28 comparable to that reported for the S. mansoni molecule. The nucleotide and deduced amino acid sequences of cDNA clones corresponding to Sj28 and Sm28 were compared. Despite obvious homology (77% identity), differences were found in regions known to contain T epitopes in the S. mansoni protein which may be an explanation for the striking differences in immunogenicity in regard to antibody production in mice. The 26-kDa GSTs of these two parasites (Sj26 and Sm26) are also closely related on the basis of nucleotide and deduced amino acid sequences, there being 82% identity in the putative coding regions. When the amino acid sequences of Sj28 and Sm28 were compared with those of Sj26 and Sm26, the overall sequence identity was approximately 20%. However, a relatively conserved region was identified in otherwise structurally different molecules which may participate in common properties of these enzymes.     

Cloning and expression of a gene encoding Sm16, an anti-inflammatory protein from Schistosoma mansoni

The gene encoding Sm16, an anti-inflammatory, immunomodulatory protein present abundantly in secretions of the infective stages of Schistosoma mansoni was cloned and partially characterized. A data base analysis showed sequence homology to an earlier reported schistosomular stathmin-like gene sequences reported in dbEST and Genbank. The putative gene coding for Sm16 is of 500 bp with an open reading frame of 117 aa that included an N-terminal signal peptide sequence of 18 aa. There are three potential sites for phosphorylation (two serine and one tyrosine residue) but no glycosylation sites in the sequence. The coding region of Sm16 was amplified from cercarial cDNA, cloned and expressed in bacterial and insect expression systems. The purified recombinant protein showed strong immunoreactivity with a polyclonal rabbit anti-Sm16 antibody raised against the native anti-inflammatory protein Sm16. Contrary to earlier report, this gene appears to be not stage-specific. Metabolic labeling studies suggested that Sm16 is phosphorylated and is synthesized by both cercariae and schistosomula of S. mansoni. Sequence homology with human stathmin, a cell cycle regulatory phospho protein, was 30%. However, when probed with specific antibodies, no cross reactivity was observed between Sm16 and human stathmin.     

Cloning, molecular characterization, and functional activity of Schistosoma japonicum glyceraldehyde-3-phosphate dehydrogenase, a putative vaccine candidate against schistosomiasis japonica.

We report the cloning, molecular characterization, and purification of functionally active recombinant glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from the human bloodfluke Schistosoma japonicum. The GAPDH homolog from the related species Schistosoma mansoni has shown correlation of antibody titer to resistance to reinfection. A 1,164-bp cDNA (C1) was isolated from an S. japonicum lambda ZapII cDNA expression library immunoscreened with hyperimmune rabbit serum raised against soluble adult S. japonicum proteins. The open reading frame of C1 encodes a protein of 338 amino acids exhibiting 90% identity to the amino acid sequence of S. mansoni GAPDH. The inferred molecular mass of the protein is 36,589 daltons, and in vitro translation of the cDNA with [35S]methionine produced a radiolabelled band of the predicted size. Antibodies to C1 selected from hyperimmune rabbit serum by affinity purification recognized an S. japonicum protein doublet of 37 kDa but did not cross-react with a corresponding protein in S. mansoni extracts. The S. japonicum GADPH appears to be translated from a single mRNA encoded by a single-copy gene. After subcloning in the QIAexpress vector pQE-10 and subsequent expression, the recombinant protein was purified under nondenaturing conditions and shown to exhibit functional GAPDH enzymatic activity.     

Identification and molecular cloning of a 67-kilodalton protein in Schistosoma japonicum homologous to a family of actin-binding proteins.

A monoclonal antibody to Schistosoma japonicum which conferred significant protection against cercarial challenge in mice was produced. The predicted translation product of the cDNA corresponding to the antigen recognized by this antibody was homologous to a newly identified family of actin-binding proteins. The expressed protein bound polymerized actin and was recognized by serum from patients infected with S. japonicum.     

Further characterisation of the Schistosoma japonicum protein Sj23, a target antigen of an immunodiagnostic monoclonal antibody.

Sj23, the 23-kDa target antigen in Schistosoma japonicum adult worms of the hybridoma monoclonal antibody (mAb) I-134, has been identified and cloned from cDNA libraries, mAb I-134 has been successfully used in immunodiagnostic assays to detect S. japonicum infection in Philippine patients. Sequence analysis has shown that Sj23 is the homologue, with 84% amino acid identity, of Sm23, a 23-kDa molecule from S. mansoni worms previously described from our laboratory. The domain structures of Sj23 and Sm23 are strikingly similar to the human membrane proteins ME491, CD37, CD53 and TAPA-1, which may suggest a functional role for the schistosome molecules in cellular proliferation.     

A stage-specific calcium-binding protein expressed in eggs of Schistosoma mansoni.

A cDNA sequence encoding a Schistosoma mansoni egg antigen SmE16 was cloned in Escherichia coli. The 16-kDa polypeptide deduced from the nucleotide sequence is related to the calmodulin and troponin C gene families of calcium-binding proteins, and the most significant homology is displayed around the four calcium-binding sites. The antigen was expressed as a hybrid protein of the bacteriophage MS2 polymerase. The MS2-SmE16 fusion protein binds calcium, as demonstrated via ligand blotting with 45Calcium. The detection of antibodies to the purified recombinant egg antigen in sera of schistosomiasis patients opens up the possibility that it may be a useful candidate for the development of serodiagnostic assays. The function of the protein in the egg is presently unclear.     

日本血吸虫大陆株26kDa谷胱甘肽S┐转移酶编码区基因的克隆及序列测定

从日本血吸虫大陆株成虫分离总ＲＮＡ，逆转录合成ｃＤＮＡ第一链，根据菲律宾株２６ｋＤａ谷胱甘肽Ｓ－转移酶（ＧＳＴ）的ｃＤＮＡ序列，设计并合成引物，扩增出２６ｋＤａ的编码区基因，并克隆到ｐＢｌｕｅｓｃｒｉｐｔ质粒。初步酶切鉴定后，从两端对插入片段进行核苷酸序列测定。结果与菲律宾株比较，核苷酸同源性为９９．８％，仅第５８２位碱基不同，菲律宾株为Ａ，而大陆株为Ｇ。比较从ｃＤＮＡ推导出的氨基酸序列，两者１００％相同。测序结果也与曼氏血吸虫进行了比较。     

Molecular cloning and tissue distribution of a 26-kilodalton Schistosoma mansoni glutathione S-transferase

A Schistosoma mansoni cDNA library was constructed from the mRNA of adult worms in the expression vector lambda gt11 and screened with a rabbit antiserum raised against the 26-kDa S. mansoni glutathione S-transferase isoforms (Sm GST 26). Two clones were selected and the nucleotide sequences deduced. The predicted amino acid sequence, specified by these cDNAs, shows strong homology with a Schistosoma japonicum 26 kDa glutathione S-transferase and a lower level of homology with mammalian glutathione S-transferase class mu isoenzymes (EC 2.5.1.18). No significant homology score was found with a 28-kDa S. mansoni glutathione S-transferase (Sm GST 28). A study of the tissue distribution of the cloned Sm GST 26 by immunoelectron microscopy shows similarities to Sm GST 28 in that they are present in the tegument and in subtegumentary parenchymal cells. However, a major difference exists in the protonephridial region in which Sm GST 26 is present in the cytoplasmic digitations localized in the apical chamber delineated by the flame cell body, suggesting that Sm GST 26 may be actively excreted by adult worms.     

Screening and Primary Characterization of NewAntigen Genes of Schistosoma Japonicum

Thestudiesonanti schistosomiasisvaccineshavebeen studiedwithrapidprogressinrecentyears[1].Aseries ofantigenicgenesofSchistosomahavebeencloned,but thesevaccinecandidatescouldnotinducehighlevelsof resistanceagainstschistosomeinfection.Currentexperim entsso…     

Expression of Schistosoma japonicum antigens in Escherichia coli

A cloned library of DNA complementary to the mRNA of adult Schistosoma japonicum has been prepared and expressed as fusion proteins with Escherichia coli beta-galactosidase. Colonies expressing the S. japonicum cDNA clones were screened both with antibodies from individuals with a history of schistosomiasis and with antibodies obtained from a rabbit immunized with whole adult worms. In both cases colonies were detected which bound antibody, although the frequency of antigen-positive clones was much higher with the rabbit antiserum than with human sera. In both cases the proportion of colonies reacting with antibodies was markedly lower than that published for equivalent screens of Plasmodium falciparum cDNA with sera from individuals with a history of falciparum malaria. Several major S. japonicum antigens were identified by the affinity purification of antibodies using immobilised fusion proteins produced during lytic growth of the recombinant bacteriophage.     

The diagnostic value and molecular characterisation of an Echinococcus multilocularis antigen gene clone

A diagnostic Echinococcus multilocularis antigen gene (EM4) has been expressed using the Escherichia coli expression vector pGEX-1 resulting in the high level synthesis of a readily purifiable, soluble, non-denatured peptide fused to the 26-kDa glutathione-S-transferase of Schistosoma japonicum. This recombinant antigen, on testing by enzyme-linked immunosorbent assay (ELISA) with heterologous human antisera, demonstrated 100% E. multilocularis specificity. Specific anti-EM4 antibody immunoprecipitated a single 66-kDa protein from protoscolex total RNA directed in vitro translation products indicating the probable involvement of post-translational modification in the production of the native EM4 antigens. Southern blotting analysis suggests that the EM4 native antigens are coded for by a single-copy gene and that the genomic organisation of the EM4 related genes in other parasites is not conserved. The nucleotide sequence of the cloned EM4 cDNA molecule has been obtained and the derived amino acid sequence shows no significant homology with other existing protein sequences.     

应用EST和电子克隆策略研究血吸虫表达基因谱

目的开展血吸虫表达基因谱的研究,寻找新的疫苗候选分子和药物靶标。方法应用表达序列标签（EST）和电子克隆策略。结果获得了552个EST序列和487个电子延伸序列,其中104个EST序列在延伸前未表现同源性,而延伸后表现出有意义的同源性;获得了日本血吸虫基因表达谱的信息以及发现了有潜在药物和疫苗价值的新基因序列。结论本研究为日本血吸虫基因表达谱的研究提供更有效的研究思路。     

Molecular cloning and characterization of a mu-class glutathione S-transferase from Clonorchis sinensis

In biliary passages, Clonorchis sinensis causes epithelial hyperplasia and is assumed to promote carcinogenesis. Glutathione S-transferase (GST) is an antioxidant enzyme involved in phase II defense in trematodes. A clone (pcsGSTM1) encoding a GST was identified by screening a C. sinensis cDNA library with a PCR-synthesized cDNA probe. The predicted amino acid sequence encoded by pcsGSTM1 cDNA had a high degree of sequence identity and folding topology similar to the mu-class GSTs. The estimated molecular mass of the protein, 26 kDa, was consistent with an expression by pcsGSTM1 cDNA. The bacterially expressed recombinant csGSTM1 protein possessed an enzymatic GST activity and conjugated GSH to reactive carbonyls of lipid peroxidation. The recombinant csGSTM1 protein did not share antigenic epitope(s) with GSTs of Fasciola hepatica, Paragonimus westermani and Schistosoma japonicum. The csGSTM1 was identified to a mu-class GST in C. sinensis.     

Molecular cloning and characterization of a novel Schistosoma japonicum "irradiated vaccine-specific" antigen, Sj14-3-3.

A Schistosoma japonicum cDNA coding for a full length S. japonicum 14-3-3 protein was obtained by antibody screening of an adult worm cDNA library using sera taken from mice vaccinated with UV-attenuated cercariae, which are capable of transferring high levels of passive immunity to this parasite. The deduced amino acid sequence consists of 254 amino acids and is highly homologous with 14-3-3 family of proteins from a variety of species (55-69% identity). The recombinant S. japonicun 14-3-3 protein (rSj14-3-3) was expressed and purified in pGEX/E. coli, and in Western blotting was strongly recognised by sera from mice, rats and bovines vaccinated with irradiated S. japonicum cercariae. Analysis of mRNA showed that Sj14-3-3 is expressed in sporocysts and adult worms, but not in cercariae, however mouse antisera against rSj14-3-3 recognised a 29 kDa native antigen in antigen preparations made from eggs, cercariae, schistosomula and adult worms of S. japonicum indicating that this antigen is present in all life-cycle stages. The presence of the native antigen in detergent extracts of intact schistosomula suggests that it is also present in the schistosomular tegument which is the most vulnerable target for immune attack. However, antisera against rSj14-3-3 did not recognise a similar band in S. mansoni or S. haematobium antigens, indicating that, like the UV-attenuated vaccines, this protein induced species-specific immune responses. Southern blot analysis suggested that there may exist more than one gene copy and/or polymorphism for Sj14-3-3. Immunoelectron microscopy confirmed that the native antigen is present throughout the body of adult worms including the tegument, but is less abundant in the muscles. The potential of rSj14-3-3 as a vaccine is now under further investigation.     

An analysis of the calcium-binding protein 1 of Fasciola gigantica with a comparison to its homologs in the phylum Platyhelminthes

A full-length cDNA encoding the Fasciola gigantica calcium-binding protein 1 (FgCaBP1) was cloned from an adult stage cDNA expression library in an immunoscreen using rabbit immune serum against the parasite's excretion/secretion antigens. The deduced amino acid sequence showed 96.3% identity to Fh22CBP of Fasciola hepatica. During development in the mammalian host FgCaBP1 RNA was detected in metacercariae, juveniles and adults and was exclusively localized to the tegumental cell bodies. Immune serum of a rabbit infected with F. gigantica detected recombinant FgCaBP1 starting from the sixth week of infection. Immune sera of mice infected with Schistosoma mansoni and Schistosoma mekongi cross-reacted with recombinant FgCaBP1 in immunoblots. Recombinant FgCaBP1 showed calcium and magnesium-binding activity by a mobility shift during non-denaturing PAGE in the presence of Ca2+ or Mg2+, respectively. A polyclonal mouse anti-rFgCaBP1 antiserum detected the native protein as a major component of the parasite's tegumental antigens in immunoblots and as a strictly tegumental antigen in tissue cross-sections of adult and juvenile parasites. Comparative sequence analysis of homologs from Fasciola and Schistosoma present in the GenBank database revealed sequence signatures specific to these trematode proteins and thereby indicates their origin from a single ancestor. FgCaBP1 contains two adjacent, N-terminal located EF-hands and a C-terminal located domain similar to dynein light chain type 1. Independent structure predictions of the two domains suggest that they will fold according to the already determined structures of the EF-hand motif and the dynein light chain type 1 proteins.     

Cloning of a 21.7-kDa vaccine-dominant antigen gene of Schistosoma mansoni reveals an EF hand-like motif

Several cDNA clones encoding a 21.7-kDa antigen (Sm21.7) were detected from a Schistosoma mansoni sporocyst cDNA expression library using irradiated cercaria-vaccinated rabbit serum. The antigen was designated ‘vaccine dominant’ because parasite-derived Sm21.7 was recognised preferentially by mouse vaccine sera compared with mouse infection sera. The cDNA and corresponding gDNA sequences showed 64% identity at the nucleotide level and 47% identity at the amino acid level with the sequence of a previously described S. mansoni tegumental antigen, sma22.6 [23]. Whereas sma22.6 mRNA occurs almost exclusively in the adult worm, Sm21.7 mRNA was equally abundant in the sporocyst, schistosomular and adult stages. Both Sm21.7 and sma22.6 sequences reveal a motif strongly homologous to the EF hand calcium binding domain but lacking the invariant glycine in the calcium binding loop. The disruptive nature of the glutamine which in Sm21.7 replaces the glycine explains why the motif is non-functional, as shown by the inability of Sm21.7 to bind calcium.