OBJECTIVETo examine the expression profile and immunofluorescence localization of the major egg antigen p40 of Schistosoma japonicum (Sjp40) during granuloma formation in the liver of infected New Zealand white rabbits.

METHODSNew Zealand white rabbits were infected with S. japonicum cercariae, and the livers were harvested at 29 and 45 days post-infection (dpi). The total RNA of the liver tissues was extracted for expression profiling of Sjp40 by quantitative reverse transcription-PCR (qRT-PCR) with GAPDH of S. japonicum as the endogenous reference gene. The expression of Sjp40 in the liver were detected by Western blotting using anti-Sjp40 monoclonal antibody (mAb) 9G7 or anti-Toxoplasma gondii tSAG1 mAb Y3A8 (control) as the primary antibody. Paraffin sections of the liver were prepared for observing egg granuloma formation using HE staining and for indirect immunofluorescence assay of Sjp40 location in the trapped eggs and egg granulomas.

RESULTSThe level of Sjp40 mRNA in the eggs trapped in rabbit livers was significantly higher at 45 dpi than that at 29 dpi (P<0.05), and Western blotting confirmed the presence of Sjp40 protein in the rabbit livers at both 29 and 45 dpi. Immunofluorescence assay demonstrated localized expression of Sjp40 in the immature eggs in the rabbit liver at 29 dpi, but at 45 dpi fluorescence was detected in clusters of mature eggs containing miracidium and in the surrounding egg granulomas.

CONCLUSIONSThe transcriptional levels of Sjp40 significantly increased with the maturation of eggs trapped in the rabbit livers. Sjp40 protein spread from the eggs to the surrounding egg granuloma at 45 dpi when acute liver granulomatous lesions occur, suggesting that Sjp40 plays a key role in egg granulomas formation in the livers of infected New Zealand white rabbits.

Animals ; Antibodies, Monoclonal ; Antigens, Helminth ; metabolism ; Fluorescent Antibody Technique ; Gene Expression Profiling ; Granuloma ; parasitology ; Helminth Proteins ; metabolism ; Liver ; parasitology ; RNA, Messenger ; Rabbits ; Schistosoma japonicum ; Schistosomiasis japonica

Calcium-binding protein is an indispensable protein which performs extensive and important functions in the growth of Schistosoma japonicum. Based on our primary study on tegument surface proteins of S. japonicun, a cDNA encoding a 66 kDa calcium-binding protein of S. japonicum (Chinese strain) was cloned, sequence analysis revealed that it was identical with that of SjIrV1 of Philippines strains S. japonicum. The expression of SjIrV1 were detected by Real-time PCR, using cDNA templates isolated from 7, 14, 21, 28, 35 and 42 days worms and the results revealed that the gene was expressed in all investigated stages, and the mRNA level of SjIrV1 is much higher in 42 d female worms than that in 42 d male worms. The cDNA containing the open reading frame of IrV1 was subcloned into a pET28a (+) vector and transformed into competent Escherichia coli BL21 for expression. The recombinant protein was purified using a Ni-NTA purification system, and confirmed by high performance liquid chromatography (RP-HPLC) and tandem mass spectrometry (MS/MS). Western blotting analysis showed that recombinant SjIrV1 (rSjIrV1) could be recognized by the S. japonicum infected mouse serum and the mouse serum specific to rSjIrV1, respectively. Immunofluorescence observation exhibited that SjIrV1 was mainly distributed on the tegument of the 35-day adult worms. ELISA test revealed that IgG, IgG1 and IgG2a antibodies are significantly increased in the serum of rSjIrV1 vaccinated mice. The study suggested that rSjIrV1 might play an important role in the development of S. japonicum.
Animals ; Antibodies, Helminth ; blood ; Calcium-Binding Proteins ; genetics ; metabolism ; Cloning, Molecular ; Escherichia coli ; metabolism ; Female ; Genetic Vectors ; Helminth Proteins ; genetics ; metabolism ; Male ; Mice ; Recombinant Proteins ; genetics ; metabolism ; Schistosoma japonicum ; genetics ; metabolism

Phosphoglycerate mutase (PGAM) is a key enzyme in glycolytic pathways. With PCR technique based on an EST identified in our lab, a novel gene named SjPGAM (GenBank Accession No. EU374631) was cloned. Sequence analysis revealed that the ORF of SjPGAM gene contained 753 nucleotides, encoding 250 amino acids, and the molecular weight was about 28.26 kD. Real-time PCR analysis showed that the mRNA level of SjPGAM was much higher in the 14 days and 19 days schistosomula than other stages, suggesting that the gene was a schistosomula stage differential expression gene. The SjPGAM cDNA fragment was subcloned into an expression vector pET-28a (+) and transformed into Escherichia coli BL21 cells. In the presence of IPTG, the 31 kD fusion protein was expressed in included bodies. Western blotting revealed that the fusion protein could be recognized by the rabbit serum anti-Schistosoma japonicum adult worm antigen preparation. The study provides important basis for investigating the mechanism of the PGAM in the glycolytic pathways of Schistosoma japonnicum.
Animals ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Male ; Phosphoglycerate Mutase ; genetics ; immunology ; RNA, Messenger ; genetics ; metabolism ; Rabbits ; Recombinant Proteins ; genetics ; metabolism ; Schistosoma japonicum ; enzymology ; genetics ; Schistosomiasis japonica ; immunology ; parasitology

To construct a hepatic stellate cells (HSCs) subtracted cDNA library to find differentially expressed genes in normal mice and mice infected with Schistosoma japonicum (S. japonicum). Suppression subtractive hybridization (SSH) was used. The cDNA fragments of normal mouse were compared to those of schistosoma-infected mice to find differentially expressed genes. Then differentially expressed cDNA fragments were directly inserted into T/A cloning vector to set up the subtractive library. Amplification of the library was carried out with transformation of DH5alpha. The amplified library contained more than 400 positive bacterial clone, which were then hybridized with forward and backward subtracted probes for differential screening. One hundred positive bacterial clones were randomly selected for sequencing and BLAST analysis. Finally, virtual Northern Blot confirmed such differential expression. The subtracted cDNA library of differentially expressed genes of HSCs was constructed successfully, the library is efficient and lays foundation for screening and cloning new and specific genes of schistosomiasis.
Animals ; Base Sequence ; Blotting, Northern ; DNA, Complementary ; genetics ; Gene Expression Regulation ; genetics ; Gene Library ; Hepatocytes ; metabolism ; Liver ; cytology ; metabolism ; Male ; Mice ; Mice, Inbred BALB C ; RNA, Messenger ; analysis ; genetics ; Schistosoma japonicum ; Schistosomiasis japonica ; genetics

For further research of the apoptosis mechanism of Schistosoma japonicum (S. japonicum). The cDNA encoding Sjcaspase3 of Schistosoma japonicum was amplified by polymerase chain reaction (PCR) technique, which contained 900 nucleotides and encoded 299 amino acids. The theory molecular weight and isoelectric point (PI) of the deduced protein is 33.5 kDa and 6.39, respectively. Real-time PCR was used to analyze the transcription profiles of Sjcaspase3 at different development stages of S. japonicum. The results showed that this gene was expressed in all stages of S. japonicum with the highest expression in 21d worms, and the level of gene transcription in 42 d female worms was higher than that of male worms. The recombinant plasmid pXJ40-FLAG-Sjcaspase3 was constructed and transfection into Hela cells successfully. Real-time PCR and Western blotting analysis showed Sjcaspase3 was successfully expressed in Hela cells. Enzyme activity analysis revealed that recombinant Sjcaspase3 possessed the activity to cut substrate DEVD. Flow cytometry proved that Sjcaspase3 could induce early apoptosis of Hela cells. The results provide the basis for proceeding further study on the biological function of Sjcaspase3 and better understand the apoptosis mechanism of S. japonicum.
Animals ; Apoptosis ; Blotting, Western ; Caspase 3 ; genetics ; metabolism ; Cloning, Molecular ; DNA, Complementary ; Female ; HeLa Cells ; Helminth Proteins ; genetics ; metabolism ; Humans ; Male ; Real-Time Polymerase Chain Reaction ; Recombinant Proteins ; Schistosoma japonicum

OBJECTIVE: To clone the full-length gene encoding succinate dehydrogenase iron-sulfur protein of Schistosoma japonicum (SjSDISP) Chinese strain and express it in Escherichia coli. METHODS: According to the published incomplete EST (BU804141) of SjSDISP and the sequence of multiclone sites of lambda gt11 vector, 2 pairs of primers were designed and synthesized. Then the 3' and 5'ends of the EST of the SjSDISP from adult Schistosoma japonicum cDNA library were amplified by anchored PCR. After sequencing, a full-length cDNA sequence of the SjSDISP was obtained, and then it was cloned into prokaryotic expression vector pGEX-4T-1. Identified by agarosed gel electrophoresis, endonucleases digestion and PCR, the resultant recombinant plasmid was used for the expression under the temperature-dependent condition and Western blot analysis. RESULTS: A 1,071 bp sequence was obtained. Sequence analysis showed that the fragment contained a complete open reading frame (ORF), encoding 278 amino acid residues. This target fragment was cloned into the prokaryotic expression vector pGEX-4T-1, and expressed in Escherichia coli. SDS-PAGE revealed that the molecular weight of the expressed fusion recombinant product was 56 kD. Western blot showed that the recombinant protein was recognized by polyclonal rabbit antiserum immunized with Schistosoma japonicum adult worm antigen. CONCLUSION Cloning of the full-length gene encoding SjSDISP and its bacterial expression were successfully done.
Amino Acid Sequence ; Animals ; Base Sequence ; Cloning, Molecular ; Escherichia coli ; metabolism ; Helminth Proteins ; biosynthesis ; genetics ; Iron-Sulfur Proteins ; biosynthesis ; genetics ; Molecular Sequence Data ; Recombinant Proteins ; biosynthesis ; genetics ; Schistosoma japonicum ; genetics ; metabolism ; Sequence Homology ; Succinate Dehydrogenase ; biosynthesis ; genetics

BACKGROUNDType 2 cytokine interleukin (IL)-13 and its decoy receptor, IL-13 receptor (R) alpha2 appear to play a major role in tissue fibrosis of schistosomiasis and asthma. IL-13 is a key regulator of the extracellular matrix (ECM). It is known to signal to cells by binding to the IL-13Ralpha1, which then heterodimerizes with IL-4Ralpha. In contrast, IL-13Ralpha2 binds IL-13 with high affinity but does not signal. IL-13Ralpha2 is known to down-regulate granulomatous inflammation and prolong host survival in Schistosoma mansoni (S. mansoni) infection, but little is known about the location and expression level of IL-13Ralpha2 in the context of S. japonicum infection.

METHODSWe established S. japonicum-infected mouse models. Kinetic serum levels of IL-13Ralpha2 were examined with ELISA. IL-13Ralpha2 mRNA and protein of liver tissues were determined by PCR and immunoblotting analysis, respectively. Detection of IL-13Ralpha2 expression and location in macrophages was performed by TaqMan PCR and fluorescent immunocytochemistry technique, respectively.

RESULTSA marked elevation of mRNA and protein expression of IL-13Ralpha2 was observed in mice during S. japonicum infection. An enhanced expression of IL-13Ralpha2 was further demonstrated in primary macrophages of murine schistosomiasis.

CONCLUSIONSIL-13Ralpha2 in macrophages may be a critical contributor to pathogenesis of schistosomiasis. The data highlight the potential importance of cell signaling and antifibrotic gene therapeutics in T helper 2 cell (Th2)-mediated diseases.

Animals ; Blotting, Western ; Cells, Cultured ; Enzyme-Linked Immunosorbent Assay ; Female ; Interleukin-13 Receptor alpha2 Subunit ; metabolism ; Macrophages ; immunology ; Male ; Mice ; Mice, Inbred BALB C ; Random Allocation ; Reverse Transcriptase Polymerase Chain Reaction ; Schistosoma japonicum ; pathogenicity ; Schistosomiasis japonica ; immunology ; microbiology

OBJECTIVETo explore therapeutic effect of Haobieyangyinruanjianfang (HBYYRJ) on mouse liver fibrosis by schistosomiasis.

METHODSMice except for normal control were infected with Japanese schistosome cercarias, after 12 weeks, infected mice were divided into 7 groups: low HBYYRJ group, middle HBYYRJ group, high HBYYRJ group, Fufangbiejiaruangan tablet (FFBJRG) group, colchicine group, 3 months infection group and 6 months infection group. Hepatic fibrosis was found in 3 months infection group. Liver hydroxyproline (Hyp) was determined, matrix metalloproteinase-2 and 9 (MMP-2, MMP-9) were detected with gelatin zymography, serum hyaluronic acid (HA) and precollagen III (PC-III) were detected using RIA.

RESULTSHBYYRJ obviously reduced hepatic fibrosis (probability value less than 0.01). Collagen and HA in 3 months infection group and 6 months infection group were higher than that in normal group (probability value less than 0.01), collagen in high and middle HBYYRJ groups and HA in middle and low HBYYRJ groups were lower than that in 6 months infection group (P less than 0.01, probability value less than 0.05). The expression of MMP-9 and MMP-2 in 3 months infection group and 6 months infection group was higher than that in normal group (probability value less than 0.01), The expression of MMP-9 in three HBYYRJ groups and the expression of MMP-2 in high HBYYRJ group were lower than that in 6 months infection group (probability value less than 0.05).

CONCLUSIONHBYYRJ can reduce liver fibrosis caused by schistosomiasis.

Animals ; Collagen Type III ; blood ; Disease Models, Animal ; Drugs, Chinese Herbal ; isolation & purification ; pharmacology ; therapeutic use ; Female ; Hyaluronic Acid ; blood ; Hydroxyproline ; metabolism ; Liver ; drug effects ; metabolism ; pathology ; Liver Cirrhosis, Experimental ; drug therapy ; etiology ; metabolism ; pathology ; Male ; Materia Medica ; isolation & purification ; pharmacology ; therapeutic use ; Matrix Metalloproteinase 2 ; metabolism ; Matrix Metalloproteinase 9 ; metabolism ; Mice ; Schistosoma japonicum ; Schistosomiasis japonica ; complications ; Severity of Illness Index ; Sex Factors ; Treatment Outcome

The 26S proteasome is a proteolytic complex responsible for the degradation of the vast majority of eukaryotic proteins. Regulated proteolysis by the proteasome is thought to influence cell cycle progression, transcriptional control, and other critical cellular processes. A novel Schistosoma japonicum gene (GenBank Accession No. AY813725) proteasome alpha2 subunit (SjPSMA2) was cloned. Sequence analysis revealed that the ORF of SjPSMA2 gene contains 708 nucleotides encoding 235 amino acids, and the molecular weight was estimated to be 25.84 kDa. Real-time PCR analysis showed that this gene expressed in 7 d, 13 d, 18 d, 23 d, 32 d and 42 d schistosoma. The mRNA level of SjPSMA2 was lower in 7 d and 23 d schistosomulum than that in other stages. The SjPSMA2 cDNA fragment was subcloned into an expression vector pET28a(+) and transformed into E. coli BL21 (DE3) cells. After induction with IPTCQ the 30 kDa fusion protein was produced as included bodies. Western-blotting revealed that the fusion protein could be recognized by the rabbit serum anti-Schistosoma japonicum adult worm antigen preparation, and the protein in native could be detected. After immunization of BALB/c mice with the fusion protein, the reduction rates of worm counts and liver egg counts were 12.33% and 35.23%. ELISA results revealed that the vaccinated group showed a significant increase in the level of IgG antibody. This study provided an important basis for investigating the regulation mechanism of the proteasome during the development of Schistosoma japonicum.
Animals ; Antibodies, Helminth ; blood ; Base Sequence ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Genes, Helminth ; Helminth Proteins ; genetics ; metabolism ; Immunization ; Liver ; parasitology ; Male ; Mice ; Mice, Inbred BALB C ; Molecular Sequence Data ; Parasite Egg Count ; Proteasome Endopeptidase Complex ; biosynthesis ; genetics ; immunology ; Rabbits ; Recombinant Proteins ; biosynthesis ; genetics ; immunology ; Schistosoma japonicum ; genetics ; metabolism ; Vaccines, Synthetic ; immunology

Radiation sensitive protein 23 (RAD23) is a nucleotide excision repair (NER) protein that plays an important role in Ubiquitin-proteasome pathway (UPP). Schistosoma japonicum radiation sensitive protein23 (SjRAD23) cDNA sequences were amplified by PCR and cloned into pET28a (+) vector to construct recombinant expression plasmid pET28a(+)-SjRAD23. The recombinant protein was expressed as both inclusion bodies and the supernatant in Escherichia coli BL21 (DE3) cell. Immunofluorescence observation shows that SjRAD23 was mainly distributed on the tegument surface of the worms. ELISA assay reveals that specific IgG, IgG1 and IgG2a antibodies could be detected in the sera of rSjRAD23 immunized mice. Western blotting analysis shows that the recombinant SjRAD23 could be recognized by serum specific to soluble adult worm antigen of S. japonicum. BALB/c mice vaccinated with rSjRAD23 combined with 206 adjuvant revealed 35.94% worm reduction and 40.59% liver egg reduction when compared with that of the adjuvant control
Animals ; Antibodies, Helminth ; blood ; Blotting, Western ; Cloning, Molecular ; DNA Repair Enzymes ; genetics ; metabolism ; DNA, Complementary ; Enzyme-Linked Immunosorbent Assay ; Escherichia coli ; Genetic Vectors ; Helminth Proteins ; genetics ; immunology ; Immunoglobulin G ; blood ; Mice ; Mice, Inbred BALB C ; Recombinant Proteins ; genetics ; immunology ; Schistosoma japonicum ; genetics ; metabolism ; Schistosomiasis japonica ; prevention & control ; Vaccines ; immunology