To identify SJCHGC01743 gene of Schistosoma japonicum and evaluate the potential of the recombinant protein as a new vaccine candidate for schistosomiasis, polymerase chain reaction (PCR) technique was used to amplify the cDNA of the gene and real-time RT-PCR was used to analyze the transcription profiles of SJCHGC01743 at different development stages. Recombinant plasmid was successfully constructed and transformed into competent Escherichia coli BL21 (DE3). Then the recombinant protein was expressed, purified and emulsified with ISA206 adjuvant to immunize BALB/c mice for three times. The immunogenicity was confirmed by Western blotting and tissue localization was detected by indirect immunofluorescent assay. The specific antibody level was detected by ELISA. The immunoprotection of rSjOST48 was evaluated by the reduction in worm and egg counts in mice. A cDNA with 1 248 nucleotides was isolated from 28-day-old schistosomes cDNAs by PCR. Sequence analysis revealed that SJCHGC01743 was a 48-kDa subunit of the oligosaccharyltransferase complex (OST48) and named as SjOST48. Real-time PCR analysis indicated that this gene was expressed in all investigated stages and had the highest expression level in 28 d worms, the level of gene transcription in female worms was significantly higher than that of male worms. Then recombinant plasmid pET28a(+)-SjOST48 was successfully constructed and expressed in E. coli BL21 (DE3). Western blotting analysis showed that rSjOST48 had good immunogenicity. Indirect immunofluorescent analysis revealed that SjOST48 was mainly distributed on the tegument of the worms. The result of ELISA indicated that the rSjOST48 vaccinated group could induce a significant increase in the level of specific IgG, IgG1 and IgG2a. An immunoprotection experiment showed that the vaccination of rSjOST48 in mice induced 32.62% (P < 0.05) reduction in the numbers of worms and 57.61% (P < 0.01) in eggs in liver, compared with that of the control group. This study provides the foundation for proceeding further research on the biological function of SjOST48 and screening new vaccine candidates for schistosomiasis.
Animals ; Antibodies, Helminth ; blood ; Cloning, Molecular ; DNA, Complementary ; Escherichia coli ; Female ; Genes, Helminth ; Helminth Proteins ; genetics ; immunology ; Immunoglobulin G ; blood ; Male ; Mice ; Mice, Inbred BALB C ; Real-Time Polymerase Chain Reaction ; Recombinant Proteins ; immunology ; Schistosoma japonicum ; genetics ; Schistosomiasis japonica ; prevention & control ; Vaccination

UNLABELLEDObjective To compare the impact of right ventricular apical (RVA) versus right ventricular outflow tract (RVOT) pacing on left ventricular systolic synchronization.

METHODSSixty patients were prospectively recruited and randomized into RVA group (n=30) with the right ventricle leads placed in the RVA and RVOT group (n=30) with right ventricle leads placed in the septum of the RVOT. Speckle tracking imaging was performed with 100% ventricle pacing to measure the differences in the time to maximum left ventricle (LV) radial strain.

RESULTSIn RVA group, the difference in the time to 6-segment maximum LV radial strain after pacing was 105.27 ± 19.74 ms, significantly greater than that in RVOT group (41.65 ± 12.17 ms, P<0.001). The standard difference of time to 6-segment maximum LV radial strain was also significantly greater in RVA group than in RVOT group (42.71 ± 17.63 vs 17.63 ± 5.62 ms, P<0.001).

CONCLUSIONLeft ventricle systolic synchronizaition after RVOT pacing is superior to RVA pacing.

Cardiac Pacing, Artificial ; methods ; Heart ; Heart Ventricles ; Humans ; Systole

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

OBJECTIVETo select and identify ssDNA aptamers specific to Streptococcus mutans strains with different cariogenicity isolated from clinical specimens.

METHODSSubtractive SELEX technology targeting the whole intact cells was used to screen for ssDNA aptamers specific to the clinical isolates Streptococcus mutans strains with different cariogenicity. Radioactive isotope, flow cytometry, gene cloning and sequencing, MEME online software and RNA structure analysis software were employed to analyze the first and secondary structures of the aptamers and identify the screened aptamers.

RESULTSDetection by radioactive isotope showed sufficient pool enrichment after 9 rounds of subtractive SELEX. Flow cytometry showed that the selected aptamers H1, H16, H4, L1, L10 and H19 were capable of binding specifically with highly cariogenic Streptococcus mutans strains but not with strains with a low cariogenicity. The aptamer H19 had the strongest binding capacity to highly cariogenic Streptococcus mutans strains, with a dissociation constant of 69.45∓38.53 nmol/L.

CONCLUSIONWe have obtained the ssDNA aptamers specific to the clinical isolates of highly cariogenic Streptococcus mutans strains.

Aptamers, Nucleotide ; genetics ; Cloning, Molecular ; DNA Primers ; Dental Caries ; microbiology ; Gene Library ; Humans ; Nucleic Acid Conformation ; SELEX Aptamer Technique ; Species Specificity ; Streptococcus mutans ; classification ; genetics ; isolation & purification

OBJECTIVETo screen of high cariogenicity Streptococcus mutans (S. mutans) strains isolated from clinical specimens preliminary.

METHODSAcidogenicity, aciduricity, extracellular polysaccharide production and adhesion of 41 strains of S. mutans isolated from clinical specimens were investigated to screen high cariogenicity S. mutans strains.

RESULTSThere were different cariogenicity among 41 strains of S. mutans, in which 3 strains of S. mutans had all high ability to produce extracellular polysaccharide, adhere to the saliva-coated hydroxyapatite, produce acid and tolerate acid, indicated there were 3 strains with high cariogenicity S. mutans strains isolated from clinical specimens. Another 3 strains of S. mutans with all low ability to produce extracellular polysaccharide, adhere to the saliva-coated hydroxyapatite, produce acid and tolerate acid indicated they were low cariogenicity S. mutans strains isolated from clinical specimens.

CONCLUSIONWe may have obtained high cariogenicity S. mutans strains isolated from clinical specimens.

Dental Caries ; Durapatite ; Humans ; Saliva ; Streptococcus mutans

OBJECTIVETo identify Streptococcus mutans (S. mutans) strains from clinical samples.

METHODSPlaque samples from caries-active and caries-free sites on enamel surfaces were obtained and cultivated for S. mutans isolation. Morphology, biochemistry, automatic microorganism analysis system and polymerase chain reaction using primers homologous to surface protein antigen I/II (spaP), glucosyltransferase B (gtfB) and dextranase (dexA) were used to identify S. mutans. Genotype of isolated S. mutans was determined by arbitrarily primed polymerase chain reaction.

RESULTSForty-six strains of S. mutans were obtained from the 32 subjects and were identified as S. mutans by biochemistry, automatic microorganism analysis system and polymerase chain reaction. Five identical genotypes were found by arbitrarily primed polymerase chain reaction.

CONCLUSIONForty-one strains of S. mutans with different genotype were obtained from clinical samples.

Dental Caries ; Dental Plaque ; Genotype ; Glucosyltransferases ; Humans ; Polymerase Chain Reaction ; Streptococcus mutans

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

The present study was intend to clone and express the cDNA encoding Cyclophilin B (CyPB) of Schistosoma japonicum, its preliminary biological function and further immunoprotective effect against schistosome infection in mice. RT-PCR technique was applied to amplify a full-length cDNA encoding protein Cyclophilin B (Sj CyPB) from schistosomula cDNA. The expression profiles of Sj CyPB were determined by Real-time PCR using the template cDNAs isolated from 7, 13, 18, 23, 32 and 42 days parasites. The cDNA containing the Open Reading Frame of CyPB was then subcloned into a pGEX-6P-1 vector and transformed into competent Escherichia coli BL21 for expressing. The recombinant protein was renaturated, purified and its antigenicity were detected by Western blotting, and the immunoprotective effect induced by recombinant Sj CyPB was evaluated in Balb/C mice. The cDNA containing the ORF of Sj CyPB was cloned with the length of 672 base pairs, encoding 223 amino acids. Real-time PCR analysis revealed that the gene had the highest expression in 18-day schistosomula, suggesting that Sj CyPB was schistosomula differentially expressed gene. The recombinant protein showed a good antigenicity detected by Western blotting. Animal experiment indicated that the vaccination of recombinant CyPB protein in mice led to 31.5% worm and 41.01% liver egg burden reduction, respectively, compared with those of the control. A full-length cDNA differentially expressed in schistosomula was obtained. The recombinant Sj CyPB protein could induce partial protection against schistosome infection.
Animals ; Antigens, Helminth ; immunology ; Cloning, Molecular ; Cyclophilins ; biosynthesis ; genetics ; immunology ; DNA, Complementary ; genetics ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; genetics ; Immunization ; Mice ; Mice, Inbred BALB C ; Recombinant Proteins ; biosynthesis ; genetics ; immunology ; Schistosoma japonicum ; genetics ; immunology ; Schistosomiasis japonica ; prevention & control ; Vaccines, Synthetic ; biosynthesis ; immunology

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

Microtus fortis is naturally resisitent to Schistosoma japonicum. In order to find schistosome-resistence-related genes of Microtus fortis, a T7 phage-display cDNA library from liver of Microtus fortis was screened with the soluble lysate of schistosomula. The specific phages were enriched 375-fold after 3 rounds of biopanning. Ninety-two positive clones picked at random were sequenced and 19 ESTs including 6 unreported genes were obtained. Compared with the negative phage clone control, five positive clones, No.4 (GenBank Accession No.: EW968294), No.13 (GenBank Accession No.: EW968303), No.14 (GenBank Accession No.: EW968304), No.15 (GenBank Accession No.: EW968305) and No.18 (GenBank Accession No.: EW968308) could induce significantly higher schistosomula mortality rate when co-cultivated with schistosomula. According to the function analysis and the shistosomula-killing effect in vitro, the genes encoding CASP8 and FADD-like apoptosis regulator isoform protein, alpha-2-HS-glycoprotein, M4 protein, R3H domain (binds single-stranded nucleic acids) isoform 2 and 3 previously unreported proteins (No.14, No.15 and No.18) obtained here, were schistosomiasis-resistence-related genes of Microtus fortis.
Animals ; Arvicolinae ; genetics ; parasitology ; Bacteriophage T7 ; genetics ; Cloning, Molecular ; Expressed Sequence Tags ; Gene Library ; Genes, Helminth ; genetics ; Immunity, Innate ; genetics ; Larva ; genetics ; growth & development ; Liver ; chemistry ; Schistosoma japonicum ; genetics ; growth & development