Schistosoma japonicum: analysis of eggshell protein genes, their expression, and comparison with similar genes from other schistosomes.

As the egg of Schistosoma japonicum plays a central role in transmission and in pathogenesis, we sought to understand the molecular biology of egg formation. In this study we characterized an eggshell protein gene of S. japonicum and compared it with similar genes from S. mansoni and S. haematobium. To initiate studies on the eggshell protein genes of S. japonicum, a cloned genomic fragment containing an entire copy of a S. haematobium eggshell protein gene was used to identify three EcoRI hybridizing fragments of 2.6, 2.0, and 1.3 kbp in S. japonicum genomic DNA and to isolate three independent genomic clones from a S. japonicum genomic library. Two genomic clones, SJ 4-1 and SJ 3-1, contain at least two copies of the gene. The DNA sequence of a 2.0-kbp EcoRI fragment of clone SJ 3-1 showed two open reading frames (ORF), one of which showed a strong homology to the chorion proteins of insects. This ORF had 207 amino acids with a calculated molecular size of 18.5 kDa. The predicted peptide was glycine (50%) and tyrosine (10%) rich like other described schistosome eggshell proteins. Primer extension and the dideoxynucleotide sequence of the mRNA defined the cap site of the RNA and positioned the putative TATA and CAAAT elements and other cis-acting elements. Northern analysis demonstrated that eggshell protein mRNA was only detected in mature female parasites. The appearance of the female-specific mRNA was dependent on pairing with the male parasite and increased with egg production (as determined by hybridization intensity). A comparison of the DNA and deduced protein sequences of eggshell protein genes from S. japonicum with those of similar genes from S. mansoni and S. haematobium indicated that the genes are highly conserved, with S. mansoni and S. haematobium genes being more similar to each other than either is to S. japonicum.     

Incorporation of radiolabelled amino acids by adult Schistosoma japonicum: further characterization of a putative eggshell precursor protein.

The synthesis patterns of female-specific proteins of Schistosoma japonicum were further investigated with particular reference to the 34 kDa putative eggshell precursor protein. Adult male and female worms of S. japonicum were metabolically labelled with 14C-tyrosine, 14C-glycine and 35S-methionine in vitro. The rates of amino acid incorporation for female worms were significantly higher than for males in all radiolabelling experiments. Labelled proteins were resolved by two-dimensional gel electrophoresis and visualized by fluorography. By using 14C-tyrosine and 14C-glycine, the 34 kDa female protein band resolved into three major spots with pI 6.0, 5.8 and 5.6. On the other hand, labelling studies using 35S-methionine failed to reveal synthesis of any corresponding spots at Mr 34 kDa. These results, together with the observations that eggshell hydrolysates are very rich in glycine but poor in methionine, suggested that the 34 kDa putative eggshell precursor protein of S. japonicum consists of at least three isoelectric forms. In addition, we have demonstrated several other female-specific polypeptides synthesized by this worm.     

Schistosoma japonicum: localization of calpain in the penetration glands and secretions of cercariae

A monoclonal antibody was generated against the large subunit of Schistosoma japonicum calpain to study the localization and possible function of the molecule in vivo. Mice were immunized with recombinant S. japonicum calpain and polyclonal antisera and a monoclonal antibody specific to schistosome calpain was obtained. In immunohistochemistry, a monoclonal antibody against S. japonicum calpain, KG-2E11, bound weakly to calpain expressed at the surface of adult worm tegument, however, it bound strongly to the cercarial secretions ("footprints") of S. japonicum, emitted from the penetration glands. The present study indicates that calpain is multifunctional as it is expressed at various locations in different developmental stages. Calpain-based vaccines could thus possibly induce protective immunity against cercariae and the following early developing stages.     

日本血吸虫SjIrV1的基因特性及免疫保护效果

钙结合蛋白是日本血吸虫生长发育不可缺少的蛋白,具有非常广泛而重要的功能.在课题组日本血吸虫体被表膜蛋白研究基础上,利用PCR技术克隆了中国大陆株日本血吸虫66 kDa钙结合蛋白(SjIrV1)编码基因的cDNA序列,BLAST分析与菲律宾株日本血吸虫SjIrV1 cDNA编码序列一致,荧光定量PCR分析表明该基因在童虫和成虫期不同发育阶段均有表达,其中在35d和42d成虫中表达量较高,在42d雌虫中该基因表达水平远高于42d雄虫.构建重组表达质粒pET28a(+)-SjIrV1,在大肠杆菌中成功诱导表达,重组蛋白主要以可溶性形式存在,通过高效液相色谱法(RP-HPLC)以及串联质谱法(MS/MS)鉴定所获蛋白为目的蛋白SjIrV1.蛋白质印迹(Western blotting)分析结果显示重组蛋白能被感染日本血吸虫鼠血清和免疫鼠血清所识别,SjIrV1蛋白在虫体各发育阶段中均表达.免疫荧光染色实验观察表明SjIrV1主要分布在日本血吸虫成虫的表膜.应用重组蛋白免疫BALB/c小鼠后,免疫鼠血清中检测到较高水平的特异性IgG、IgG1和IgG2a抗体.结果表明SjIrV1可能在日本血吸虫的生长发育过程中起着重要作用.     

Characterisation of a family of Schistosoma japonicum proteins related to dynein light chains.

Dynein light chains (DLC) are components of dynein, an enzyme complex involved in various aspects of microtubule-based motility. We report here the molecular cloning and sequencing of cDNAs encoding a family of DLC-like polypeptides (SjcDLC1-5) from the human bloodfluke Schistosoma japonicum with open reading frames of 87-104 amino acids and deduced molecular masses ranging from 10.5 to 12.3 kDa. Two-dimensional Western blot analysis confirmed the presence of several S. japonicum DLC isoforms with differing pI values and molecular sizes. We also describe the molecular characterisation, genomic organisation and expression of clone SjcDLC1, and the immunological characterisation and localisation of its encoded protein. Northern blot analysis of adult worm RNA indicated SjcDLC1 is encoded by a single message of approximately 650 bp and Southern analysis suggested one SjcDLC1 gene exists in the S. japonicum genome. Immunolocalisation studies demonstrated that the SjcDLC1 protein is present in the tegument of the adult and cercarial stages of S. japonicum. SjcDLC1 and the other SjcDLC may function in the transport of specialised organelles, comprising membranous and discoid bodies, through the tegument to the schistosome-unique heptalaminate tegumental membrane at the external surface of the adult worm. As a consequence, they may provide novel targets for anti-schistosome vaccine and/or drug development.     

Femcoat, a novel eggshell protein in Drosophila: functional analysis by double stranded RNA interference.

In Drosophila oogenesis, follicle cells derived from somatic tissue surround the oocyte and play key roles in generating properly polarized oocytes. During the later steps of oogenesis, follicle cells are involved in secretion of proteins that make the eggshell, an essential protective layer for the oocyte. Although studies on the signaling processes to make polarized oocytes have been progressed very far, studies on the mechanisms for eggshell formation is not clear yet. To elucidate the underlying mechanism in eggshell formation, we used a differential display screen to isolate genes that are specifically expressed during the later stages of oogenesis, and isolated a novel gene, Femcoat. Femcoat encodes a putative chorion membrane protein that contains many highly charged residues and has a putative signal peptide. Femcoat is expressed specifically in the follicle cells with a punctate staining pattern typical of secreted proteins, and becomes cross-linked heavily at the final steps of oogenesis. To identify the developmental role of Femcoat in eggshell formation, we performed an inducible double stranded RNA mediated interference (dsRNAi) method to specifically reduce Femcoat expression during oogenesis in adult flies. Electron microscopy analysis of egg chambers from these flies showed defects in chorion formation. These pieces of evidence demonstrated that Femcoat is necessary for eggshell formation, especially during chorion synthesis. Our results demonstrate that inducible dsRNAi analysis can be effective in determining the developmental function of novel genes.     

Detection of inducible nitric oxide synthase in Schistosoma japonicum and S. mansoni

Schistosoma japonicum and S. mansoni were tested for reactivity with an anti-inducible nitric oxide (iNOS) antibody and the distribution of iNOS was studied by immunofluorescent tests in different stages of the parasites. Reactivity was associated with the tegument in both larval schistosomes (sporocysts and cercariae) and eggs. With adult worms, the majority of the immunofluorescence was predominantly subtegumental in S. japonicum and parenchymal in S. mansoni. Fluorescence was also observed in host tissues (snails and mouse liver). In Western blots, the enzyme of S. japonicum had an apparent molecular weight of about 210 kDa. The possible role of worm and host iNOS in the parasite-host interrelation remains to be clarified.     

Schistosoma Japonicum UDP-Glucose 4-Epimerase Protein Is Located on the Tegument and Induces Moderate Protection against Challenge Infection

Schistosomiasis is an important global public health problem, as millions of people are at risk of acquiring this infection. An ideal method for sustainable control of schistosomiasis is using a vaccine alone or in combination with drugs. In the present study, we cloned the SjGALE gene and generated the expression product in E. coli. The expression level of SjGALE during different developmental stages of S. japonicum was evaluated by real-time RT-PCR and western blotting. Immunolocalization indicated that the protein was mainly located on the tegument of the parasite. Infection of rSjGALE-immunized mice demonstrated a 34% and 49% reduction of the mean worm burden and liver egg burden, respectively, in two independent experiments, indicating immune protection. The liver egg count from each female adult worm was significantly reduced by 63% in the two trials. The cytokine profile and IgG isotype analysis demonstrated the induction of a Th1 immune profile in response to immunization with this protein, further suggesting protection against infection. In conclusion, these findings indicated that SjGALE is a potential vaccine against S. japonicum.     

Hox genes from the parasitic flatworm Schistosoma japonicum

Hox genes are characterized by a highly conserved peptide domain and contribute to antero-posterior axis patterning during embryogenesis. These genes have been widely studied in a variety of animal species due to their central role in evolutionary developmental biology. Based on the published genome assembly and unpublished re-sequencing project data, we present the first genome-wide characterization and comparative genomic analysis of the Hox gene family within Schistosoma japonicum. Eight Hox genes were identified and validated in our investigation. Phylogenetic analysis revealed that these genes are distributed among seven orthology groups of the Hox gene family. Our study further suggested that differences in the Lox5 gene copy number existed between the two closely related species, S. japonicum and Schistosoma mansoni. Semi-quantitative real-time polymerase chain reaction experiments revealed that Lox5 and Hox4 gene expression was high in the schistosomulum stage, and all four genes investigated showed highest expression within the eggs.     

Isolation and characterization of surface antigens from Schistosoma mansoni. II. Antigenicity of radiolabeled proteins from adult worms.

Adult Schistosoma mansoni were radiolabeled in vitro with 125I Bolton-Hunter reagent. Surface membrane antigens were solubilized with non-ionic detergent, then reacted with infection or normal serum. The antigen-antibody complexes were then precipitated with staphylococcal protein A immunoadsorbent, eluted with urea and SDS, and fractionated by SDS-PAGE. The results indicated the presence of 6 to 8 tegument antigens, depending on the type of antisera used. Human antisera to S. japonicum and S. haematobium reacted with some but not all of the antigens identified with human S. mansoni infection serum; this implies the presence of species-specific tegument antigens. The molecular weights of the radiolabeled antigens ranged from 10,000 to 100,000. A large (greater than 100,000) molecular weight glycoprotein and an uncharacterized lipid fraction appeared to be precipitated nonspecifically. Immunoprecipitation methods with anti-mouse IgG and anti-mouse whole serum failed to detect the presence of hostlike antigens in the labeled extracts. Several of the labeled proteins from S. mansoni were found to react with serum from patients infected with either S. haematobium or with S. japonicum.     

日本血吸虫凋亡基因Sjcaspase3的克隆、真核表达 及其功能分析

为深入研究日本血吸虫细胞凋亡机制。利用PCR技术扩增得到Sjcaspase3的全长序列,其ORF含900 bp,编码299个氨基酸,理论分子量为33 509.7 Da,理论等电点为6.39。Real-time PCR分析表明该基因在日本血吸虫生长发育的各个时期均有表达,其中21 d表达量最高,42 d雌虫表达量高于42 d雄虫。成功构建了p XJ40-FLAG-Sjcaspase3重组质粒并转染到Hela细胞内,荧光定量PCR和Western blotting分析表明Sjcaspase3成功在Hela细胞中表达。酶活分析提示重组Sjcaspase3具有切割特异性底物天冬氨酸-谷氨酸-缬氨酸-天冬氨酸(DEVD)的活性。流式细胞术检测了Sjcaspase3可诱导Hela细胞发生早期细胞凋亡。研究结果为深入探讨Sjcaspase3的生物学功能及日本血吸虫细胞凋亡机制奠定了基础。     

Cloning and characterization of a Schistosoma mansoni 1H and 30S clones as two tegumental vaccine candidate antigens

Two Schistosoma mansoni cDNA clones 30S and 1H were identified by immunoscreening of sporocyst lambdagt11 library and by random sequencing of clones from lambdaZap libraries, respectively. Clone 30S was one of 30 clones identified by an antibody raised against tegument of 3-h schistosomules. The clone was found to encode an 81 amino-acid protein fragment. It was expressed in Escherichia coli as a fusion protein of calculated molecular mass of about 35 kDa with C-terminus of Schistosoma japonicum glutathione-S-transferase (Sj26; about 26 kDa). The recombinant fusion protein was specifically recognized by serum of rabbits immunized with irradiated cercariae. Clone 1H is one of 76 expressed sequence tags derived from an adult worm library. It encodes the complete sequence of a tegumental membrane protein, Sm13. The 104 amino-acid open reading frame encodes a protein with a calculated molecular mass of about 11.9 kDa. Clone 1H was expressed in E. coli as an insoluble fusion protein with Sj26 of about 40 kDa. In Western blots, the fusion protein was recognized by serum from rabbits vaccinated with irradiated cercariae but not by preimmune rabbit sera. The cloning, characterization and expression of those proteins are therefore potentially usefull for vaccine development.     

日本血吸虫SjRAD23基因的克隆表达及基因重组抗原的免疫保护效果

辐射敏感蛋白23具有核苷酸切除修复功能,在泛素蛋白酶体途径中起到重要作用。本研究利用PCR技术克隆了日本血吸虫辐射敏感蛋白23(Sj RAD23)编码的c DNA序列,成功获得Sj RAD23的基因序列,其ORF为1 053 bp。构建Sj RAD23基因重组表达质粒p ET28a(+)-Sj RAD23,并在大肠杆菌BL21中成功诱导表达,重组蛋白在上清和沉淀中都有存在。利用免疫组化技术检测该蛋白在虫体的分布情况,该蛋白广泛分布在日本血吸虫虫体被膜。用重组蛋白免疫BALB/c小鼠后,免疫小鼠血清中检测到较高水平的特异性Ig G、Ig G1和Ig G2a。Western blotting分析显示重组蛋白能够被日本血吸虫成虫可溶性抗原免疫小鼠血清所识别。用重组蛋白r Sj RAD23免疫结果与206佐剂对照组比较,r Sj RAD23在BALB/c小鼠中诱导了35.94%减虫率,40.59%肝脏减卵率。结果表明Sj RAD23具有作为疫苗候选分子的潜力。