TSO18 gene was subcloned into the Pichia pastoris expression vector pPIC9K. The recombinant plasmid pPIC9K-TSO18 was transformed into P. pastoris GS115 by electroporation so that the plasmid will be integrated with chromosome of P. pastoris. The P. pastoris strains containing multi-copy recombinant were screened by G418 and induced by methanol. The expression product was analyzed by SDS-PAGE, Western blot, deglycosylation, and purified by Sephadex column, and was used to immunize mice. The results indicated that the target protein was efficiently expressed in P. pastoris, and glycosylated moderately, and had immunological activity. In a 5 liter fermentor, the expression level of the target protein was up to 2.54 mg/mL. These results will benefit for the development of genetically engineering vaccine.
Animals ; Antigens, Helminth ; biosynthesis ; genetics ; immunology ; Cloning, Molecular ; Electroporation ; Gene Expression ; Genetic Vectors ; genetics ; Mice ; Pichia ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; immunology ; Swine ; Taenia solium ; genetics ; immunology

OBJECTIVETo clone the coding gene of the stage-specific antigen cC1 from Cysticercus cellulosae and express high levels of soluble cC1 in E.coli.

METHODSThe cC1 gene was amplified from Cysticercus cellulosae by RT-PCR and cloned into pMD18-T vector, followed by subcloning into the prokaryotic expression plasmid pET28a. The recombinant plasmid was transformed into E.coli BL21(DE3) and the expression conditions were optimized. The expressed product was purified by Ni(+)-affinity chromatography, analyzed by high-performance liquid chromatography (HPLC), and identified with SDS-PAGE and Western blotting.

RESULTSThe fragment length of the amplification product by RT-PCR was 1056 bp. Comparison of the amplified gene sequence with the cC1 gene in Genbank identified a samesense point mutation at 423 position in the gene cloned into the expression plasmids. After a 6-h induction with 0.05 mmol/L IPTG at 37 degrees celsius;, the expression of the 40 kd soluble fusion protein exceeded 60% of the total bacterial protein, and the fusion protein was recognized by Cysticercus-infected human sera. The purity of the fusion protein was about 94% after purification by affinity chromatography.

CONCLUSIONThe stage-specific antigen cC1 from Cysticercus cellulosae has been successfully cloned and the soluble protein efficiently expressed in E.coli, which provides the basis for its further study and application.

Animals ; Antigens, Helminth ; biosynthesis ; genetics ; immunology ; Cloning, Molecular ; Cysticercus ; immunology ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; Humans ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology ; Solubility ; Swine ; Taenia solium ; immunology

To obtain the recombinant 18 kD protein with high purity and normal bioactivity of Cysticercus cellulosae (rCE18), E. coli cells with the rCE18 were disrupted ultra-sonically, and the inclusion bodies were washed with a solution containing 0.2% deoxycholic acid sodium (DOC)and 2% DOC, respectively. Then they were denatured with 0.9% sodium lauroyl sarcosine (SKL) followed by dialysis and gel filtration to refold and purify the target protein. At the same time, this method was compared with GST-FF affinity chromatography and recovering from SDS-PAGE gel. Biological activity of purified rCE18 was analyzed with indirect ELISA, and the purity of the products was identified using SDS-PAGE. The purity of refolded inclusion bodies exceeded 60% and the total recovery of activated protein rCE18 was about 41.3%. The specificity of rCE18 reached up to 97.2% using indirect ELISA. An effective way for purifying and refolding rCE18 expressed in E. coli as inclusion bodies was established, rCE18 with higher purity and activity was obtained, which has the potential for developing diagnosis methods of porcine cysticercosis.
Animals ; Antigens, Helminth ; biosynthesis ; genetics ; immunology ; isolation & purification ; Chromatography, Gel ; Cysticercus ; genetics ; immunology ; metabolism ; Escherichia coli ; genetics ; metabolism ; Inclusion Bodies ; metabolism ; Protein Renaturation ; Recombinant Proteins ; genetics ; immunology ; isolation & purification

The mammalian trematode Paragonimus westermani is a typical digenetic parasite, which can cause paragonimiasis in humans. Host tissues and blood cells are important sources of nutrients for development, growth and reproduction of P. westermani. In this study, a cDNA clone encoding a 47 kDa hemoglobinase of P. westermani was characterized by sequencing analysis, and its localization was investigated immunohistochemically. The phylogenetic tree prepared based on the hemoglobinase gene showed high homology with hemoglobinases of Fasciola hepatica and Schistosoma spp. Moreover, recombinant P. westermani hemoglobinase degradaded human hemoglobin at acidic pH (from 3.0 to 5.5) and its activity was almost completely inhibited by E-64, a cysteine proteinase inhibitor. Immunohistochemical studies showed that P. westermani hemoglobinase was localized in the epithelium of the adult worm intestine implying that the protein has a specific function. These observations suggest that hemoglobinase may act as a digestive enzyme for acquisition of nutrients from host hemoglobin. Further investigations may provide insights into hemoglobin catabolism in P. westermani.
Sequence Alignment ; Recombinant Proteins/biosynthesis/genetics ; Phylogeny ; Paragonimus westermani/*enzymology/genetics ; Molecular Sequence Data ; Hemoglobins/metabolism ; Escherichia coli/enzymology/genetics ; DNA, Complementary/genetics ; Cysteine Endopeptidases/*genetics/immunology/*metabolism ; Astacoidea/parasitology ; Antigens, Helminth/genetics/immunology/metabolism ; Animals ; Amino Acid Sequence

In December 2011, we reported an autochthonous case of Echinococcus multilocularis infection in a 42-year-old woman in Korea. The diagnosis was based on histopathological findings of the surgically resected liver cyst. In the present study, we evaluated the serological and molecular characteristics of this Korean E. multilocularis case. The patient's serum strongly reacted with affinity-purified native Em18 and recombinant Em18 antigens (specific for E. multilocularis) but negative for recombinant antigen B8/1 (reactive for Echinococcus granulosus). In immunoaffinity chromatography, the serum also strongly reacted with E. multilocularis and only weakly positive for E. granulosus. We determined the whole nucleotide sequence of cox1 (1,608 bp) using the paraffin-embedded cystic tissue which was compared with E. multilocularis isolates from China, Japan, Kazakhstan, Austria, France, and Slovakia. The Korean case showed 99.8-99.9% similarity with isolates from Asia (the highest similarity with an isolate from Sichuan, China), whereas the similarity with European isolates ranged from 99.5 to 99.6%.
Adult ; Animals ; Antibodies, Helminth/*blood ; Antigens, Helminth/genetics/*immunology/metabolism ; Base Sequence ; Echinococcosis, Hepatic/*immunology/parasitology ; Echinococcosis, Pulmonary/diagnosis/genetics/immunology ; Echinococcus granulosus/genetics/immunology ; Echinococcus multilocularis/genetics/*immunology/isolation & purification ; Electron Transport Complex IV/genetics ; Female ; Humans ; Mitochondria/genetics ; Molecular Sequence Data ; Republic of Korea ; Sequence Analysis, DNA

Dendritic cells (DCs), which are regarded as the most potent antigen-presenting cells, are involved in innate and adaptive immunity. Upon uptake of pathogens, DCs express cell surface markers and secrete cytokines. In this study, we analyzed production of cytokines and found that interleukin (IL)-10 and transforming growth factor (TGF)-beta production significantly increased in bone marrow-derived DCs and a mouse DC line, DC2.4, after treatment with crude antigen (CA) from liver fluke, Clonorchis sinensis. However, expression patterns of several activation molecules did not change. In addition, following treatment of DC2.4 cells with antigen from the lung fluke, Paragonimus westermani, production of IL-10 and TGF-beta significantly increased compared with groups treated with other parasite antigens, Spirometra erinacei plerocercoid CA and Echinococcus granulosus hydatid cystic fluid. We also found that treatment of DC2.4 cells with C. sinensis CA resulted in rapid and significant phosphorylation of extracellular signal-regulated kinase 1/2, a mitogen-activated protein kinase. Following treatment of DC2.4 cells with C. sinensis CA, treatment with an inhibitor specific to an extracellular signal-regulated kinase inhibited production of IL-10 and TGF-beta. Our results suggest that CA from C. sinensis has a role in the anti-inflammatory function of DC cells by inducing IL-10 and TGF-beta through activation of extracellular signal-regulated kinase 1/2.
Animals ; Antigens, Helminth/*pharmacology ; Cells, Cultured ; Clonorchis sinensis/*immunology ; Dendritic Cells/drug effects/*metabolism ; Interleukin-10/genetics/*metabolism ; MAP Kinase Signaling System ; Mice ; Transforming Growth Factor beta/genetics/*metabolism

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

Infection of schistosomiasis japonica may eventually lead to liver fibrosis, and no effective antifibrotic therapies are available but liver transplantation. Hedgehog (HH) signaling pathway has been involved in the process and is a promising target for treating liver fibrosis. This study aimed to explore the effects of pentoxifylline (PTX) on liver fibrosis induced by schistosoma japonicum infection by inhibiting the HH signaling pathway. Phorbol12-myristate13-acetate (PMA) was used to induce human acute mononuclear leukemia cells THP-1 to differentiate into macrophages. The THP-1-derived macrophages were stimulated by soluble egg antigen (SEA), and the culture supernatants were collected for detection of activation of macrophages. Cell Counting Kit-8 (CCK-8) was used to detect the cytotoxicity of the culture supernatant and PTX on the LX-2 cells. The LX-2 cells were administered with activated culture supernatant from macrophages and(or) PTX to detect the transforming growth factor-β gene expression. The mRNA expression of shh and gli-1, key parts in HH signaling pathway, was detected. The mRNA expression of shh and gli-1 was increased in LX-2 cells treated with activated macrophages-derived culture supernatant, suggesting HH signaling pathway may play a key role in the activation process of hepatic stellate cells (HSCs). The expression of these genes decreased in LX-2 cells co-cultured with both activated macrophages-derived culture supernatant and PTX, indicating PTX could suppress the activation process of HSCs. In conclusion, these data provide evidence that PTX prevents liver fibrogenesis in vitro by the suppression of HH signaling pathway.
Animals ; Antigens, Helminth ; isolation & purification ; pharmacology ; Cell Culture Techniques ; Cell Differentiation ; drug effects ; Cell Line ; Culture Media, Conditioned ; chemistry ; pharmacology ; Gene Expression Regulation ; Hedgehog Proteins ; agonists ; antagonists & inhibitors ; genetics ; immunology ; Hepatic Stellate Cells ; cytology ; drug effects ; metabolism ; Humans ; Liver Cirrhosis ; metabolism ; parasitology ; prevention & control ; Macrophage Activation ; drug effects ; Macrophages ; cytology ; drug effects ; immunology ; Models, Biological ; Monocytes ; cytology ; drug effects ; metabolism ; Pentoxifylline ; pharmacology ; Phosphodiesterase Inhibitors ; pharmacology ; RNA, Messenger ; genetics ; immunology ; Schistosoma japonicum ; chemistry ; Signal Transduction ; Tetradecanoylphorbol Acetate ; pharmacology ; Zinc Finger Protein GLI1 ; genetics ; immunology ; Zygote ; chemistry