No abstract available.
Animals ; *Base Sequence ; DNA, Helminth/*genetics ; DNA, Mitochondrial/*genetics ; *Databases, Nucleic Acid ; Electron Transport Complex IV/*genetics ; Helminth Proteins/*genetics

BACKGROUNDCystic echinococcosis due to Echinococcus granulosus (E. granulosus) is one of the most important chronic helminthic diseases, especially in sheep/cattle-raising regions. The larval stage of the parasite forms a cyst that grows in the liver, lung, or other organs of the host. To ensure a long life in the host tissues, the parasite establishes complex inter-cellular communication systems between its host to allow its differentiation toward each larval stage. Recent studies have reported that this communication is associated with the extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase cascade in helminth parasites, and in particular that these protein kinases might serve as effective targets for a novel chemotherapy for cystic echinococcosis. The aim of the present study investigated the biological function of a novel ERK ortholog from E. granulosus, EgERK.

METHODSDNA encoding EgERK was isolated from protoscolices of E. granulosus and analyzed using the LA Taq polymerase chain reaction (PCR) approach and bioinformatics. Reverse transcription PCR (RT-PCR) was used to determine the transcription level of the gene at two different larval tissues. Western blotting was used to detect levels of EgERK protein. The expression profile of EgERK in protoscolices was examined by immunofluorescence.

RESULTSWe cloned the entire Egerk genomic locus from E. granulosus. In addition, two alternatively spliced transcripts of Egerk, Egerk-A, and Egerk-B were identified. Egerk-A was found to constitutively expressed at the transcriptional and protein levels in two different larval tissues (cyst membranes and protoscolices). Egerk-A was expressed in the tegumental structures, hooklets, and suckers and in the tissue surrounding the rostellum of E. granulosus protoscolices.

CONCLUSIONSWe have cloned the genomic DNA of a novel ERK ortholog from E. granulosus, EgERK (GenBank ID HQ585923), and found that it is constitutively expressed in cyst membrane and protoscolex. These findings will be useful in further study of the biological functions of the gene in the growth and development of Echinococcus and will contribute to research on novel anti-echinococcosis drug targets.

Animals ; Blotting, Western ; Computational Biology ; DNA, Helminth ; genetics ; Echinococcus granulosus ; enzymology ; genetics ; Genome, Helminth ; genetics ; Helminth Proteins ; genetics ; metabolism ; Polymerase Chain Reaction

The tapeworm Taenia solium is an important human zoonotic parasite that causes great economic loss and also endangers public health. At present, an effective vaccine that will prevent infection and chemotherapy without any side effect remains to be developed. In this study, codon usage patterns in the T. solium genome were examined through 8,484 protein-coding genes. Neutrality analysis showed that T. solium had a narrow GC distribution, and a significant correlation was observed between GC12 and GC3. Examination of an NC (ENC vs GC3s)-plot showed a few genes on or close to the expected curve, but the majority of points with low-ENC (the effective number of codons) values were detected below the expected curve, suggesting that mutational bias plays a major role in shaping codon usage. The Parity Rule 2 plot (PR2) analysis showed that GC and AT were not used proportionally. We also identified 26 optimal codons in the T. solium genome, all of which ended with either a G or C residue. These optimal codons in the T. solium genome are likely consistent with tRNAs that are highly expressed in the cell, suggesting that mutational and translational selection forces are probably driving factors of codon usage bias in the T. solium genome.
Animals ; Base Sequence ; Codon/*genetics ; Evolution, Molecular ; *Genome, Helminth ; Helminth Proteins/*genetics ; Molecular Sequence Data ; Taenia solium/*genetics

Diphyllobothrium nihonkaiense has been reported in Korea as Diphyllobothrium latum because of their close morphologic resemblance. We have identified a human case of D. nihonkaiense infection using the mitochondrial cytochrome c oxidase subunit I (cox1) gene sequence analysis. On 18 February 2012, a patient who had consumed raw fish a month earlier visited our outpatient clinic with a long tapeworm parasite excreted in the feces. The body of the segmented worm was 2 m long and divided into the scolex (head) and proglottids. It was morphologically close to D. nihonkaiense and D. latum. The cox1 gene analysis showed 99.4% (340/342 bp) homology with D. nihonkaiense but only 91.8% (314/342 bp) homology with D. latum. The present study suggested that the Diphyllobothrium spp. infection in Korea should be analyzed with specific DNA sequence for an accurate species identification.
Animals ; Cyclooxygenase 1/*genetics ; Diphyllobothriasis/*parasitology ; Diphyllobothrium/enzymology/genetics/*isolation & purification ; Female ; Helminth Proteins/*genetics ; Humans ; Mitochondrial Proteins/*genetics

To express recombinant Ancylostoma caninum anticoagulant peptide-c2 (AcAPc2), a whole cDNA fragment encoding AcAPc2 was achieved by ligation- PCR and inserted into prokaryotic expression vector pTWIN1 for constructing the specific self-splicing prokaryotic expression vector, pTWIN1-AcAPc2; positive recombinants were transformed into E. coli ER2566 for expression research. The recombinant protein, AcAPc2-intein2-CBD, was soluble and expressed in E. coli ER2566 (about 30.1% fusion protein in total protein). AcAPc2-intein2-CBD was characterized to be 41 KD by SDS-PAGE and identified by Western-blot. The recombinant fusion protein was purified to a efficiently high degree by chitin affinity chromatography. After the process of specific self-splicing induced by beta-Mercaptoethanol, the target protein, AcAPc2, was obtained, characterized to be 21 KD by SDS-PAGE and migrated as a dimmer. Molecular weight of AcAPc2 conformed to native dimmer. Bio-information analysis indicated relationship between secondary construction of AcAPc2 and biologic function. These findings greatly facilitate the purification of AcAPc2 and are very important for the additional studies on its anti-coagulation mechanism and its clinical application as anti-coagulation medicine.
Animals ; Dogs ; Escherichia coli ; genetics ; metabolism ; Gene Expression ; Genes, Helminth ; Genetic Vectors ; Helminth Proteins ; biosynthesis ; genetics ; Plasmids ; genetics ; Prokaryotic Cells ; metabolism ; RNA Splicing ; Recombinant Fusion Proteins ; chemistry ; pharmacology

During the past several decades, researches on parasite genetics have progressed from biochemical and serodiagnostic studies to protein chemistry, molecular biology, and functional gene studies. Nowadays, bioinformatics, genomics, and proteomics approaches are being applied by Korean parasitology researchers. As for Clonorchis sinensis, investigations have been carried out to identify its functional genes using forward and reverse genetic approaches and to characterize the biochemical and biological properties of its gene products. The authors review the proteins of cloned genes, which include antigenic proteins, physiologic and metabolic enzymes, and the gene expression profile of Clonorchis sinensis.
Animals ; Clonorchiasis/parasitology ; Clonorchis sinensis/enzymology/*genetics/*metabolism ; Gene Expression Regulation ; Helminth Proteins/*genetics/*metabolism ; Humans

OBJECTIVETo prepare Sj14-3-3 DNA vaccine and observe its immunoprotection against Schistosoma japonicum in mice.

METHODSThe Sj14-3-3 gene was amplified by reverse transcription-polymerase chain reaction (RT-PCR) and subcloned into eukaryotic expression vector pBK. The recombinant plasmid pBK-Sj14-3-3 was extracted, purified and inoculated into BALB/c mice by intramuscular injection. Mice were attacked by Schistosoma japonicum cercariae and then killed. Adult worm and egg were counted, respectively. Diameter of the egg granulomas in the liver of infected mice was measured.

RESULTSElectrophoresis on 1% agarose gel showed that the product of RT-PCR and the inserted fragment of recombinant plasmid digested with EcoR I and Xho I had the same size, about 765 bp, confirming the latter was the 14-3-3 encoding gene by nucleotide sequencing. Adult worm load declined by 27%, average egg load of per gram (EPG) of the liver tissues by 79%, average egg production per couple of adult worm (EPWP) by 51%, and mean diameter of egg granulomas by 29% in vaccinated mice.

CONCLUSIONThe recombinant plasmid pBK-Sj14-3-3 was successfully constructed, which had some immunoprotection against Schistosoma japonicum in infected mice, indicating its potential to be vaccine candidate molecule of Schistosoma japonicum.

14-3-3 Proteins ; genetics ; immunology ; Animals ; Antibodies, Helminth ; blood ; Antigens, Helminth ; genetics ; immunology ; Cloning, Molecular ; DNA, Helminth ; genetics ; Female ; Helminth Proteins ; genetics ; immunology ; Membrane Proteins ; genetics ; immunology ; Mice ; Mice, Inbred BALB C ; Parasite Egg Count ; Rabbits ; Recombinant Proteins ; biosynthesis ; genetics ; immunology ; Schistosoma japonicum ; genetics ; immunology ; Schistosomiasis japonica ; immunology ; prevention & control ; Vaccines, DNA ; immunology

OBJECTIVETo discuss the optimal immunization dose by observing the immunoprotective effects of different doses of recombinant Schistosoma japonicum (Chinese strain) signaling protein 14-3-3 (rSj14-3-3).

METHODSSj14-3-3 gene was amplified by reverse transcriptase PCR (RT-PCR), subcloned into prokaryotic expression vector pET28a, then transformed into E.coli to express by inducing. Purified rSj14-3-3 was prepared through SDS polyacrylamide gel electrophoresis (SDS-PAGE), electroelution, dialysis, then BALB/c mice were divided into 5 groups and immunized in rSj14-3-3 protein followed by challenging infection (the 1st, 2nd, and 3rd groups were immunized in 50 microg, 100 microg and 300 microg antigen, respectively. The 4th, 5th groups were immunized in Freund's adjuvant and normal saline controls). After 6 weeks of challenging infection, the mice were killed and the worm and egg reduction rates were calculated. And the mice sera in different time were taken to examine the specific anti-Sj14-3-3 IgG.

RESULTSrSj14-3-3 protein was expressed successfully. After immunizing and challenging, worm reduction was found to be 28.20% in the 1st group, 43.10% in the 2nd group, 40.00% in the 3rd group, respectively. Number of eggs in liver tissue was reduced by 41.80%, 57.50%, 55.70%, respectively. Compared the results of the tested groups to the controls, the differences were of significance by t-test (worm reduction rate: t = 6.8 in the 1st group, t = 8.7 in the 2nd group, t = 7.3 in the 3rd group, P < 0.01 in all tested groups. Egg reduction rate at the group's number above: t = 11.23, t = 11.54, t = 7.99, P < 0.01 in all tested groups). As compared the results between the tested groups by chi(2), the differences were of significance between the 1st and the 2nd groups (worm reduction rate: chi(2) = 8.96, P < 0.05; egg reduction rate: chi(2) = 15.69, P < 0.05), between the 1st and the 3rd groups, the differences were also of significance (worm reduction rate: chi(2) = 6.52, P < 0.05; egg reduction rate: chi(2) = 12.52, P < 0.05). The difference was not of significance between the 2nd and the 3rd groups (worm reduction rate: chi(2) = 1.20, P > 0.05; egg reduction rate: chi(2) = 0.93, P > 0.05). In all tested groups, total anti-Sj14-3-3 specific IgG rose markedly. IgG(1) and IgG(2a) subtypes were high, but IgG(2b) and IgG(3) were near the background in four subtypes tested.

CONCLUSIONImmunoprotection of rSj14-3-3 should have some relations with immunization dose, and the protection obtained from immunizing mice by using 100 microg antigen was the best.

14-3-3 Proteins ; administration & dosage ; immunology ; Animals ; Antibodies, Helminth ; immunology ; Antibody Formation ; Antigens, Helminth ; blood ; Female ; Helminth Proteins ; immunology ; Immunoglobulin G ; blood ; Mice ; Mice, Inbred BALB C ; Recombinant Proteins ; Schistosoma japonicum ; genetics ; immunology ; Signal Transduction ; Vaccination

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 express and purify Schistosoma japonicum ribosomal protein S4(SjRPS4) in Escherichia coli, and assess its value in immunodiagnosis of Schistosomiasis japonica.

METHODSGene fragment of SjRPS4 was amplified by screening the cercaria cDNA library of Schistosoma japonicum. The target gene was cloned into the expressive vector pQE30 and transformed into E. coli M15. The recombinant protein expression was induced by isopropylthio-β-D-galactoside (IPTG). This fusion protein was purified by Ni(2+)-NTA chromatography and identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), Western blot and ELISA.

RESULTSThe plasmid pQE30/SjRPS4 was constructed successfully and expressed a SjRPS4 fusion protein in E. coli as showing a single special band on SDS-PAGE gel at Mr 30 × 10(3) position. It reached a purity of above 90% after purification. The Western blot result confirmed that the recombinant protein could specifically react with the serum samples from patients of schistosomiasis. Detecting the serum of Schistosomiasis japonica patients by ELISA, the sensitivity and specificity of the ELISA method were 90.91% (70/77) and 92.59% (25/27), the positive rate of recombinant protein expression was 67.30% (70/104). There was no cross-reaction with paragonimiasis patients' serum.

CONCLUSIONProtein SjRPS4 was successfully cloned and expressed, and it was confirmed that SjRPS4 antibodies were valuable in the diagnosis of Schistosomiasis japonica.

Amino Acid Sequence ; Animals ; Antibodies, Helminth ; blood ; Antigens, Helminth ; Cloning, Molecular ; Enzyme-Linked Immunosorbent Assay ; Gene Library ; Humans ; Molecular Sequence Data ; Plasmids ; Recombinant Proteins ; genetics ; Ribosomal Proteins ; genetics ; Schistosoma japonicum ; genetics ; Schistosomiasis japonica ; diagnosis ; genetics ; Sensitivity and Specificity