RESUMEN
Food-borne parasitic diseases are exhibiting new epidemiological characteristics in such society that is filled with economic development, ecological environmental changes, more frequent population flow, as well as diversities of dietary source and style. The food-borne parasitic diseases have become a major risk factor for food safety and health care, and a global public health problem. In this review, we will give an overview on the epidemiological information of some major food-borne parasitic diseases both in China and in the world, and summarize their emerging characteristics and epidemiological trends. Research on the prevention techniques and pathogenesis of the diseases is reviewed as well. Finally, perspectives are given on the diagnosis/detection, basic mechanisms of the diseases, and the strategies for prevention and transmission interruption.
Asunto(s)
Parasitología de Alimentos , Enfermedades Parasitarias , Alimentos , HumanosRESUMEN
OBJECTIVE: To construct recombinant plasmid pSPPcGT which contains signal peptide peptidase gene of Plasmodium falciparum (PJSPP) and GFP, and transfect into P. falciparum (3D7 strain) to obtain mutant parasites which can express PJSPP-GFP. METHODS: Plasmodium falciparum(3D7 strain) genomic DNA was extracted from cultured malaria parasites. The C-terminal region of PJSPP, an 883 bp gene fragment was amplified by PCR, and then cloned into pPM2GT vector to get recombinant vector pSPPcGT. The recombinant vectors were identified by PCR, double restriction enzyme digestion and DNA sequencing. pSPPcGT vector was transfected into malaria parasites. The positive clones were selected by adding inhibitor of Plasmodium falciparum dihydrofolate reductase WR99210 to the culture medium. The pSPP-GFP-transfected parasites were fixed with methanol, stained with DAPI, and observed under immunofluorescence microscope. The PJSPP-GFP expression in P. falciparum was identified by SDS-PAGE and Western blotting. RESULTS: The C-terminal region of PJSPP was amplified from P.falciparum (3D7 strain) genomic DNA by PCR with the length of 883 bp. The constructed recombinant vectors were identified by PCR screening, double restriction enzyme digestion and DNA sequencing. The pSPPcGT vector was transfected into P. falciparum and the positive clones were selected by WR99210. GFP fluorescence was observed in transfected parasites by immunofluorescence microscopy, and mainly located in the cytoplasm. The PJSPP-GFP expression in malaria parasites was confirmed by Western blotting with a relative molecular mass of Mr 64,000. CONCLUSION: Recombinant vector PJSPP-GFP is constructed and transfected into P. falciparum to obtain P. falciparum mutant clone which can express PfSPP-GFP.
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Ácido Aspártico Endopeptidasas/metabolismo , Plasmodium falciparum , Animales , Ácido Aspártico Endopeptidasas/genética , Secuencia de Bases , Electroforesis en Gel de Poliacrilamida , Vectores Genéticos , Mutación , Reacción en Cadena de la Polimerasa , TransfecciónRESUMEN
Angiostrongylus cantonensis is a rodent nematode. Adult worms of A. cantonensis live in the pulmonary arteries of rats. Humans and mice are accidental hosts or named nonpermissive hosts. The larva cannot develop into an adult worm and only causes serious eosinophilic meningitis or meningoencephalitis if humans or mice eat food containing larva of A. cantonensis in the third stage. The differing consequences largely depend on differing immune responses of the host to parasite during A. cantonensis invasion and development. Microglia is considered to be the key immune cell in the central nervous system like macrophage. To further understand the reasons for why mice and rats attain different outcomes in A. cantonensis infection, we set up the method to isolate and culture newborn rats' primary microglia and observe the activation of the microglia cells, comparing with mice microglia cell line N9. We treated cells with soluble antigen of the fourth larva of A. cantonensis (L4 larva) and measured mRNA levels of IL-1ß, IL-5, IL-6, IL-13, eotaxin, iNOS, and TNF-α by real-time PCR. The results showed that N9 expressed high mRNA level of IL-6, IL-1ß, TNF-α, iNOS, IL-5, IL-13, and eotaxin, but primary microglia only had IL-5, IL-13, and eotaxin mRNA level. It implies that microglia from rats and mice had different reaction to soluble antigen of A. cantonensis. Therefore, we supposed that microglia may play an immune modulation role during the brain inflammation induced by A. cantonensis.
Asunto(s)
Angiostrongylus cantonensis/inmunología , Antígenos Helmínticos/inmunología , Microglía/inmunología , Microglía/parasitología , Animales , Antígenos Helmínticos/aislamiento & purificación , Células Cultivadas , Citocinas/biosíntesis , Perfilación de la Expresión Génica , Larva/inmunología , Ratones , Óxido Nítrico Sintasa de Tipo II/biosíntesis , Ratas , Ratas Sprague-Dawley , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Galectin plays an important role in host-parasite interactions. In this study, we identified a novel gene encoding galectin-10 (AcGal-10) from the cDNA library of Angiostrongylus cantonensis and characterized its biological role in the parasite. Sequence and phylogeny analysis showed that AcGal-10 is related to other galectin family members with the conserved loci (H(84)-D(86)-R(88)-V(96)-N(98)-W(105)-E(108)-R(110)). The mRNA level of AcGal-10 was expressed in reactive oxygen stress radicals. We have identified two proteins of A. cantonensis galectin-10 gene, one of which was reported (AcGAL10-W) and the others is AcGAL-10-M. In addition, recombinant AcGal-10 (rAcGal-10) was constructed into the pGEX-4T-1 plasmid, purified, and finally confirmed by SDS-PAGE and LC-MS. Hemagglutination assay showed that the minimum concentration of rAcGAL10-W and rAcGAL10-M required for the hemagglutination of BALB/c mice erythrocyte was 25 µg/mL, and the carbohydrate-binding ability showed no difference between rAcGAL10-W and rAcGAL10-M. The mRNA levels of AcGal-10 were indeed expressed higher after stimulation with H(2)O(2) and recombinant A. cantonensis galectin-10. A mutation of AcGal-10 was also found, but there was no significant difference compared with the wild type. Furthermore, we also confirmed that recombinant AcGal-10 plays a role in the activation of the microglia. In conclusion, the report here showed that AcGal-10 may be an important molecule related to infection of A. cantonensis.
Asunto(s)
Angiostrongylus cantonensis/efectos de los fármacos , Angiostrongylus cantonensis/fisiología , Galectinas/biosíntesis , Perfilación de la Expresión Génica , Estrés Oxidativo , Especies Reactivas de Oxígeno/toxicidad , Secuencia de Aminoácidos , Animales , Eritrocitos/efectos de los fármacos , Galectinas/genética , Hemaglutinación , Ratones , Ratones Endogámicos BALB C , Modelos Moleculares , Datos de Secuencia Molecular , Filogenia , Unión Proteica , Conformación Proteica , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Ratas , Ratas Sprague-Dawley , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
Angiostrongylus cantonensis infection causes eosinophilic meningitis in humans. Baicalein is a flavonoid originally isolated from the roots of Scutellaria baicalensis Georgi. In this study we evaluated the efficacy of the combination of albendazole and baicalein for treating eosinophilic meningitis in BALB/c mice. Therapeutic efficacy included the survival time, body weight, neurological function, leucocyte and eosinophil counts, eotaxin concentration, matrix metalloproteinase-9 (MMP-9) activity, larval recovery and histopathological examination. The results showed that the combination of albendazole and baicalein was more effective than either drug administered singly. Combination therapy increased the survival time, decreased body weight loss, neurological dysfunction, leucocyte response, eotaxin concentration and MMP-9 activity. Our results suggest that the combination of albendazole and baicalein may exhibit synergistic beneficial effects in the treatment of eosinophilic meningitis induced by A. cantonensis.
Asunto(s)
Albendazol/uso terapéutico , Angiostrongylus cantonensis/efectos de los fármacos , Antinematodos/uso terapéutico , Flavanonas/uso terapéutico , Meningitis/tratamiento farmacológico , Infecciones por Strongylida/tratamiento farmacológico , Albendazol/administración & dosificación , Angiostrongylus cantonensis/patogenicidad , Animales , Antinematodos/administración & dosificación , Peso Corporal , Quimiocina CCL11 , Quimioterapia Combinada , Eosinófilos/citología , Flavanonas/administración & dosificación , Larva/efectos de los fármacos , Recuento de Leucocitos , Masculino , Metaloproteinasa 9 de la Matriz/metabolismo , Meningitis/mortalidad , Meningitis/parasitología , Ratones , Infecciones por Strongylida/mortalidad , Infecciones por Strongylida/parasitología , Resultado del TratamientoRESUMEN
OBJECTIVE: To study the protective immunity induced by recombinant vaccination of Cs-Rho GTPase of Clonorchis sinensis (Cs). METHODS: 20 SD-rats(8 weeks) were divided into two groups: A (recombinant protein experiment group) and B (PBS control group). Rats in group A were immunized with 1 ml protein of Cs-Rho GTPase (90 microg/ml) and 1 ml Freund's complete adjuvant through back and vola. 2 week later, the rats were given 1 ml protein of Cs-Rho GTPase (90 microg/ml) and 1 ml Freund's incomplete adjuvant, followed by 1 ml protein of Cs-Rho GTPase (90 microg/ml) through intraperitoneal injection at 4, 7, 11 week after the first immunization. Rats in group B were given PBS in the same way as group A. All rats were challenged each with 50 Clonorchis sinensis metacercariae after the last immunization. 21 d later, fecal samples were collected from all rats for examining eggs (number of eggs per gram feces, EPG) in every 3-5 d. When eggs were found, the rats were sacrificed and worms were collected. IgG, IgG1 and IgG2a in sera were detected by ELISA before every immunization. Mean number of worms and eggs, and antibody level in the experiment group were calculated and statistically compared with the controls. RESULTS: The mean number of worms and EPG were (9.2 +/- 9.9) and (956.8 +/- 1 062.5) respectively in group A, which were significantly lower than those of group B [(23.25 +/- 15.75) and (3 062.5 +/- 2 501.8) respectively] (P < 0.05). The absorbency values of serum IgG (0.1, 0.45, 0.65, 0.6, 0.65), IgG1 (0.1, 0.45, 1.1, 1.0, 1.1), and IgG2a (0.1, 0.7, 1.1, 1.1, 1.1) before every immunization in group A were significantly higher than those of group B (almost always 0.1) (P < 0.05). CONCLUSION: Recombinant vaccination of Cs-Rho GTPase induces partial protective immunity against Clonorchis sinensis infection in rats.
Asunto(s)
Antígenos Helmínticos/inmunología , Clonorquiasis/prevención & control , Vacunas/inmunología , Proteínas de Unión al GTP rho/inmunología , Animales , Anticuerpos Antihelmínticos/sangre , Anticuerpos Antihelmínticos/inmunología , Clonorchis sinensis/inmunología , Femenino , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Masculino , Recuento de Huevos de Parásitos , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes/inmunologíaRESUMEN
OBJECTIVE: To investigate the subcellular localization of ATP synthase b subunit from Clonorchis sinensis under different conditions of Hela cell cycling, and the effect of this protein on the expression of its encoding-gene and homologous host genes. METHODS: pEGFP-N1-CsATP-synt_B and the vector pEGFP-N1 were transfected into Hela cells, respectively. Transfected cells were synchronized in G0/G1 by serum starvation, G1/S, S cells by double thymidine block, and G2/M cells by thymidine-Nocodozale block. After synchronization, the subcellular localization of the expressed fusion protein was observed with a laser confocal microscope. The expression level of this fusion protein in cells was detected by flow cytometry (FCM). The expression of CsATP-synt_B and HomoATP-synt_B in different cell cycle phases accessed by RT-PCR. RESULTS: FCM results indicated that in the G0/G1 phase the expression of pEGFP-N1 vector was decreased significantly, while pEGFP-N1-CsATP-synt_B expression showed an upward trend. In the other phases of cell cycle, the protein expression was similar in the above two kinds of plasmids. The intact CsATP-synt_B was expressed in mitochondria in the G0/G1, S, and G2/M phases and nucleus during G1/S phase. After the fusion proteins entered the nucleus, the mRNA expression of CsATP-synt_B and HomoATP-synt_B increased significantly. CONCLUSION: CsATP-synt_B can be expressed in the nucleus during G1/S phase, and regulated by the cell cycle and energy requirements.
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Ciclo Celular , Núcleo Celular/metabolismo , Clonorchis sinensis/citología , Clonorchis sinensis/enzimología , ATPasas de Translocación de Protón Mitocondriales/metabolismo , Animales , Citometría de Flujo , Células HeLa , Humanos , Datos de Secuencia Molecular , Subunidades de Proteína/metabolismoRESUMEN
Saliva has been suggested as an easily accessible and a noninvasive diagnostic alternative for detection of antibodies. To identify and characterize Schistosoma japonicum (S. japonicum) antigens that are recognized by saliva of infected host, we have used a pool of saliva from infected patients to immunoscreen an egg cDNA library of S. japonicum. The open reading frame of the isolated two clones encodes same protein of 116 amino acids exhibiting 100% identity to an amino acid sequence (AY222893) of S. japonicum in NCBInr database. The protein encoded is inferred a secretory protein with a molecular mass of 13 kDa (Sj13) and shares no homology to any entries in the NCBInr database, demonstrating that Sj13 might be a schistosome-specific protein. Reverse transcriptase polymerase chain reaction, Western blotting, and immunolocalization analysis revealed Sj13 could be detected in cercaria, adult, and egg and was localized to forehead and tegument of cercaria, cell body ("cytons") of adult worm, egg shell, and epidermal plate of miracidium. Furthermore, Sj13 showed a good antigenicity when reacted with saliva or serum from schistosomiasis patients. The recombinant Sj13 (rSj13) expressed and purified from Escherichia coli was applied to detect its specific salivary antibody for schistosomiasis diagnosis by an indirect enzyme linked immunosorbent assay (ELISA). Preliminary laboratory test of 116 subjects, 40 with parasitologically proven S. japonicumm infection, 46 with other infectious diseases, and 30 negative controls exhibited 92.50% sensitivity with saliva/rSj13 and 95.00% with serum/SWAP (P > 0.05). The specificity of the ELISA using saliva/rSj13 was 92.11% versus 85.53% with serum/SWAP (P < 0.05). No direct correlations of anti-Sj13 IgG levels with egg counts in stool were observed in saliva detection. These results suggest that Sj13 specific salivary antibody detection may be useful as an antigen for the salivary diagnosis of schistosomiasis japonica and contribute to epidemiological study of schistosomiasis infection in endemic areas.
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Anticuerpos Antihelmínticos/análisis , Antígenos Helmínticos , Ensayo de Inmunoadsorción Enzimática/métodos , Saliva/química , Schistosoma japonicum/genética , Schistosoma japonicum/inmunología , Esquistosomiasis Japónica/diagnóstico , Estructuras Animales/química , Animales , Antígenos Helmínticos/química , Antígenos Helmínticos/genética , Clonación Molecular , Escherichia coli/genética , Humanos , Ratones , Peso Molecular , Sistemas de Lectura Abierta , Óvulo/química , Schistosoma japonicum/química , Sensibilidad y Especificidad , Homología de Secuencia de AminoácidoRESUMEN
Schistosomiasis is considered the most important human helminthiasis in terms of morbidity and mortality. In this study, comparative soluble proteomic analysis of normal cercariae and ultraviolet-irradiated attenuated cercariae (UVAC) from Schistosoma japonicum were carried out in view of the high efficiency of irradiation-attenuated cercariae vaccine. The results revealed that some proteins showed significant differential expression in the parasite after treatment with ultraviolet light. Total 20 protein spots were identified by mass spectrometry, corresponded to five groups according to their functions in the main that were structural and motor proteins (actin, et al.), energy metabolism associated enzymes (glyceraldehydes-3-phosphage dehydrogenase, et al.), signaling transduction pathway-associated molecules (14-3-3 protein, et al.), heat shock protein families (HSP 70 family, et al.), and other functional proteins (20S proteasome). Furthermore, our results indicated that the differential expression of the proteins by ultraviolet irradiation may be, at least partially, acquired by regulating the mRNA levels of corresponding proteins. These results may provide new clues for further exploring the mechanism of protective immunity induced by UVAC and may shed some light on the development of vaccines against schistosomiasis.
Asunto(s)
Proteínas del Helminto/análisis , Proteoma/análisis , Schistosoma japonicum/química , Schistosoma japonicum/efectos de la radiación , Rayos Ultravioleta , Animales , Electroforesis en Gel Bidimensional , Perfilación de la Expresión Génica , Espectrometría de MasasRESUMEN
OBJECTIVE: To establish and maintain the life cycle of Clonorchis sinensis in laboratory. METHODS: Adult worms and eggs of Clonorchis sinensis were collected from naturally infected cats. Eggs were ingested by freshwater snails in aquarium. When the cercariae were released from infected snails, they invaded into freshwater fishes. From the 30th day on after the release of cercariae, the infection rate and metacercariae density in freshwater fishes were determined. RESULTS: After 95 days the infected snails began shedding cercariae in a temperature range of 24.3 -37.2 degrees C, and no cercariae were found under 20 degrees C. The infection rate in the snails Parafossarulus striatulus and Alocinma longicornis was 12.5% and 18.0%, respectively. Metacercariae were found in fish at 30 days after cercariae infection, and matured metacercariae were detected in 45 days. The number of metacercariae per gram of fish meat in Pseudorasbora para, Ctenopharyngodon idellus, Rhodeus sinensis, Hypophthalmichthys nobilis, Cirrhinus molitorella, Carassius auratus, Cyprinus carpio and Oreochromis niloticus was 1 792, 16, 8, 6, 5, 4, 4, and 2, respectively. Rats and cats were fed with metacercariae from fish to receive adult worms. CONCLUSION: Life cycle of Clonorchis sinensis has been established and maintained in the laboratory.
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Clonorchis sinensis/crecimiento & desarrollo , Estadios del Ciclo de Vida , Animales , Gatos , Clonorquiasis/veterinaria , Peces/parasitología , Ratas , Caracoles/parasitologíaRESUMEN
OBJECTIVE: To illustrate the distribution of ATP synthase b subunit in the tissue of Clonorchis sinensis adult and its subcellular mimical localization in HeLa cells. METHODS: With the antiserum against recombinant CsATP-synt_B protein raised from SD rats as primary antibody, paraffin sections of the adult of C. sinensis were processed by the method of fluorescent immunohistochemistry to observe the distribution of CsATP-synt_B protein in adult worm. According to the prediction by bioinformatics of the definite mitochondrial targeting sequence (MTS) and probable Bipartite nuclear localization signals (NLS_BP)in CsATP-syntB sequence, recombinant pEGFP-N1 plasmids containing the intact and three defective CsATP-synt_B sequence with single defect of MTS or NLS_BP or double defect respectively were constructed. The recombinant plasmids and the control plasmid-pEGFP-N1, pEYFP-Mito and H2B-CFP, were transfected into the HeLa cells by Lipofectamine 2000 reagent and the subcellular location of the GFP fusion protein was observed with confocal microscopy. RESULTS: The CsATP-synt_B protein appeared to distribute all over the adult worm, especially abundant on the acetabulum, ovary, vitellarium and tegument. The intact CsATP-synt_B was definitely expressed in mitochondria and/or nucleus of infected HeLa cells, whereas the MTS-deleted mutant only in cytoplasma and nucleus, the NLS_BP-deleted mutant in mitochondria and cytoplasm, and the double defect mutant only in cytoplasm. CONCLUSION: The distribution of CsATP-synt_B in adult is accord with that of mitochondria, and mainly exits in the organs and the tissues of active energy metabolism. This study first predicted and confirmed that CsATP-synt_B can be expressed in the nucleus.
Asunto(s)
Clonorchis sinensis/enzimología , Clonorchis sinensis/metabolismo , ATPasas de Translocación de Protón/metabolismo , Animales , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Células HeLa/parasitología , Humanos , Mitocondrias/metabolismoRESUMEN
OBJECTIVE: To express enolase gene of Taenia asiatica, investigate the immunoreactivity of the recombinant TaENO protein, and immuno-histo-localize the presence of the recombinant TaENO in adults of T. asiatica. METHODS: The gene encoding enolase of T. asiatica (TaENO) was cloned by high throughput sequencing from the cDNA library of adult T. asiatica. The coding region of TaENO was amplified by PCR, and cloned into a prokaryotic expression vector pET-30a (+). The recombinant plasmid was transformed into E. coli BL-21/DE3 and followed by expression of the protein induced by IPTG. The protein was purified by Ni-IDA affinity chromatography, and tested by SDS-PAGE. Its immunoreactivity was examined by Western blotting. The mice were immunized subcutaneously with purified TaENO formulated in Freund's adjuvant. Serum samples were collected and analyzed for specific antibodies by ELISA. The localization of TaENO in adult worms was demonstrated by immunofluorescent technique. RESULTS: The recombinant expression plasmid was identified by PCR, double endonuclease digestion and sequencing. The recombinant TaENO was about Mr 47 000 with a concentration of 0.37 mg/ml. It was recognized by antisera of SD rats immunized with TaENO, sera of taeniasis patients and sera of infected swine. The immunofluorescence assay revealed that TaENO immune serum located in the tegument of T. asiatica adult. CONCLUSION: The TaENO gene has been expressed with immunoreactivity, and the recombinant TaENO is immunolocalized in the tegument of T. asiatica adult.
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Fosfopiruvato Hidratasa/genética , Fosfopiruvato Hidratasa/aislamiento & purificación , Taenia/genética , Animales , Femenino , Expresión Génica , Vectores Genéticos , Humanos , Plásmidos , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes/biosíntesis , Taenia/enzimologíaRESUMEN
Successful isolation and expansion of neural stem/progenitor cells from cynomolgus monkey (cm-NSPCs), may not only help to increase our understanding of NSPCs, but also provide an important translational tool for preclinical trials. Here we initially isolated NSPCs from aborted fetal cynomolgus monkey brain, and expanded them in adherent culture system. Then we demonstrated that cultured cm-NSPCs were almost bipolar cells proliferated in clump-like structure, expressed typical markers for NSPCs, and could differentiate into neurons, astrocytes, and oligodendrocytes. After transduction with lentivirus, 70-80% of cm-NSPCs expressed enhanced green fluorescent protein and the stemness was unaffected. This study provided basis for obtaining large numbers of cm-NSPCs, and efficient transduction of them with exogenous genes, which would facilitate cell-based therapies in nonhuman primate models, and might help to investigate the mechanism of central nervous system development and/or controlling neural regeneration.
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Feto Abortado/citología , Encéfalo/citología , Encéfalo/embriología , Macaca fascicularis/embriología , Células Madre/citología , Animales , Biomarcadores , Técnicas de Cultivo de Célula , Diferenciación Celular/genética , Linaje de la Célula/genética , Separación Celular , Células Cultivadas , Terapia Genética/métodos , Vectores Genéticos/genética , Proteínas Fluorescentes Verdes/genética , Lentivirus/genética , Regeneración Nerviosa/genética , Neuroglía/citología , Neuroglía/metabolismo , Neuronas/citología , Neuronas/metabolismo , Trasplante de Células Madre/métodos , Células Madre/metabolismo , Transfección/métodosRESUMEN
Tools from bioinformatics websites such as NCBI, ExPaSy were used for the analysis. The malate dehydrogenase full-length gene from Taenia saginata asiatica was 1 212 bp in length, with a coding region of 30-1 028 bp and coding 332 amino acids. It was a complete and full-length gene compared with the homologues in GenBank. The protein showed no transmembrane region, with stable physical-chemical characteristics. Three major linear epitopes located aa95-aa100, aa322-aa327 and aa117-aa122, with certain distance from each other on the surface of spatial structure of malate dehydrogenase (MDH). The last one was the linear epitope of Taenia. This cytosolic malate dehydrogenase gene is a potential antigen for diagnosis.
Asunto(s)
Proteínas del Helminto/genética , Malato Deshidrogenasa/genética , Taenia saginata/genética , Animales , Biología Computacional , ADN Complementario , Epítopos/genética , Datos de Secuencia Molecular , Taenia saginata/enzimología , Taenia saginata/inmunologíaRESUMEN
OBJECTIVE: To clone and express the lactate dehydrogenase (LDH) gene of Taenia saginata asiatica and analyze the immunogenicity of the recombinant protein. METHODS: By screening the full length cDNA plasmid library, the coding region of LDH was amplified with PCR, and cloned into the prokaryotic expression vector pET-30a (+), then expressed in E. coli BL21 with IPTG induction. The recombinant protein was detected by SDS-PAGE and purified by Ni-IDA affinity chromatography, and its immunogenicity was analyzed by Western blotting. RESULTS: PCR, double enzyme digestion and DNA sequencing confirmed that the recombinant expression plasmid was constructed. The expression products were obtained and purified by Ni-IDA affinity chromatography. Western blotting analysis of LDH recombinant protein testified that the recombinant protein could be recognized by sera of the Taenia saginata asiatica infected swine and the patient. CONCLUSIONS: The LDH gene of Taenia saginata asiatica has been cloned and expressed, and the purified protein has been confirmed with immunogenicity.
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Malato Deshidrogenasa/genética , Malato Deshidrogenasa/metabolismo , Porcinos/parasitología , Taenia saginata/enzimología , Animales , Clonación Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Humanos , Malato Deshidrogenasa/inmunología , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunologíaRESUMEN
OBJECTIVE: To obtain the recombinant IgE-dependent histamine-releasing factors of Schistosoma japonicum and Clonorchis sinensis (rSjHRF and rCsHRF) and to study the effect of recombinant HRFs to induce histamine release from sensitized rat mast cells. METHODS: The complete coding regions of SjHRF and CsHRF were cloned separately, and the recombinant plasmids were respectively transformed and expressed in BL21 cells. The soluble recombinant rSjHRF and rCsHRF were purified. Aliquots of the mast cells obtained from the lungs of OVA-immunized rats were separately incubated with rSjHRF and rCsHRF and the released histamine was measured by the OPT spectrofluorometric procedure. The dose-dependent curves and the kinetics of histamine release induced by rSjHRF and rCsHRF were prepared. RESULTS: The recombinant plasmids pET-30-rSjHRF and pET-30-rCsHRF were constructed successfully and the purified soluble recombinant proteins rSjHRF and rCsHRF were obtained by affinity chromatography. rSjHRF and rCsHRF induced histamine release from sensitized mast cells in a dose-dependent manner. At the concentration of 150 mg/L, the average rate of histamine release from sensitized mast cells induced by rSjHRF and rCsHRF were 49.78% and 32.63%, respectively. Histamine release increased with prolonged reaction time and the maximal release occurred at 35 min. CONCLUSION: The recombinant parasite-originated IgE-dependent HRFs show an effect of inducing histamine release from sensitized mast cells, suggesting that this protein would play a role in type I hypersensitivity in hosts with parasitic infections.
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Biomarcadores de Tumor/farmacología , Proteínas del Helminto/farmacología , Liberación de Histamina/efectos de los fármacos , Mastocitos/efectos de los fármacos , Animales , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/inmunología , Clonorchis sinensis/genética , Clonorchis sinensis/inmunología , Proteínas del Helminto/genética , Proteínas del Helminto/inmunología , Inmunoglobulina E/inmunología , Masculino , Mastocitos/metabolismo , Ratas , Ratas Wistar , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/farmacología , Schistosoma japonicum/genética , Schistosoma japonicum/inmunología , Proteína Tumoral Controlada Traslacionalmente 1RESUMEN
OBJECTIVE: To predict the structure and function of SjLDH using bioinformatics method. METHODS: By online analysis at bioinformatics websites such as NCBI (http://www.ncbi.nlm.nih.gov/) and Expasy (http://cn.expasy. org/), and employing software packages such as Vector NTI suite and PCgene to do multi-sequence homological alignment, phylogenetic analysis, secondary structure and topological prediction, homology modeling of tertiary structure, antigenic epitope analysis, etc. RESULTS: Same conservative sites and key catalytic sites existed among SjLDH and LDHs from other species. Similarity of SjLDH compared to CsLDH, TvLDH and HsLDH was 75%, 17%, 58%-60% respectively. Phylogenetic analysis demonstrated that the evolution relation between SjLDH and DmLDH was closer than the relation between SjLDH and CeLDH, the relationship between SjLDH and HsLDH-B, -C was closer than HsLDH-A. Three transmembrane regions were found, the region of 98aa-106aa in three hydrophilic regions located outside of membrane was inferred as the major antigen epitope. This antigen epitope had significant difference with LDHs from protozoon (Pf., Tg., Tv.) and had 1-3 amino acid residue difference with MmLDH, HsLDH-A, -B, -C, and was the same with CsLDH. One of the key catalytic residues and substrate (pyruvate) binding loop were located in this region. Tertiary structure demonstrated that 98aa-106aa was on the surface of the protein and formed a substrate binding loop, other two key catalytic sites were at the position near the loop. CONCLUSION: The prediction implied that LDH was an ideal molecule for phylogenetic analysis; SjLDH might be a potential molecular target for immunodiagnosis, anti-schistosome drug and vaccine development.
Asunto(s)
Biología Computacional/métodos , Proteínas del Helminto/genética , L-Lactato Deshidrogenasa/genética , Schistosoma japonicum/enzimología , Secuencia de Aminoácidos , Animales , Dominio Catalítico , Proteínas del Helminto/química , L-Lactato Deshidrogenasa/química , L-Lactato Deshidrogenasa/clasificación , Modelos Moleculares , Datos de Secuencia Molecular , Filogenia , Alineación de Secuencia/métodosRESUMEN
OBJECTIVE: To clone and express the Clonorchis sinensis F0-ATP synthase b chain (CsF0-ATP-synt_B) gene and analyze immunogenicity of the recombinant protein. METHODS: The coding region F0-ATP synthase b chain gene with the mitochondrial targeting sequence (MTS) removed was amplified with PCR using the cloned plasmid as template, and the product was cloned into the prokaryotic expression vector pET-28a(+), transformed into E. coli BL21 (DE3) and induced with IPTG. The expressed product was purified by Ni-IDA affinity chromatography,and analyzed by SDS-PAGE for its expression and identified by Western blotting for its immunogenicity. RESULTS: The coding sequence of the F0-ATP synthase b-chain like gene removed off the MTS contains 813 base pairs encoding 271 amino acids with a theoretical molecular weight of 31,171.9. PCR, double enzyme digestion and DNA sequencing confirmed that the recombinant plasmid pET-28a (+)-CsF0-ATP-synt_B was constructed successfully, and the resolvable expression was obtained in E.coli BL21. Highly purified recombinant protein was prepared through affinity chromatography. The recombinant protein could be recognized by the immune serum of the SD rat immunized with the recombinant protein. CONCLUSION: The CsF0-ATP-synt_B like gene has been efficiently expressed in prokaryotic expression system with immunogenicity.
Asunto(s)
Clonorchis sinensis/genética , Proteínas del Helminto/genética , ATPasas de Translocación de Protón/genética , Animales , Anticuerpos Antihelmínticos/sangre , Western Blotting , Clonación Molecular , Clonorchis sinensis/enzimología , ADN Complementario/química , ADN Complementario/genética , Biblioteca de Genes , Proteínas del Helminto/inmunología , Proteínas del Helminto/metabolismo , Datos de Secuencia Molecular , ATPasas de Translocación de Protón/inmunología , ATPasas de Translocación de Protón/metabolismo , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/metabolismo , Análisis de Secuencia de ADNRESUMEN
OBJECTIVE: To detect the effect of gossypol, praziquantel and artemether on activity of the recombinant lactate dehydrogenase of Schistosoma japonicum (rSjLDH). METHODS: Effect of gossypol (0-0.10 mmol/L), praziquantel (0-0.20mmol/L) and artemether (0-0.10 mmol/L) on the enzymatic activity of rSjLDH was detected by spectrophotometric method in standard reaction system established before, same solvent of each reagent was used as control. Results were analyzed by SPSS13.0 software. RESULTS: Compared with the control, considerable inhibition for both reduction and oxidation reactions catalyzed by rSjLDH was observed at 0.04 mmol/L of gossypol (P < 0.01). The enzyme activities of rSjLDH for reduction and oxidation reactions were inhibited by 91.3% and 89.1% respectively at 0.1 mmol/L of gossypol, com-pared with the control (P < 0.01). To praziquantel, enzymatic activity inhibition was observed at 0.02 mmol/L for oxidation reaction and at 0.06 mmol/L for reduction reaction (P < 0.05). At 0.2 mmol/L of praziquantel, enzymatic activities were inhibited by 83.8% for reduction reaction and 72.2% for oxidation reaction respectively, than that of the control (P < 0.01). No inhibition for both reduction and oxidation reactions was observed at 0.1 mmol/L of artemether, compared with that of the control (P > 0.05). CONCLUSION: Gossypol and praziquantel show a high inhibition on rSjLDH. SiLDH may be one of the molecular targets of praziquantel.
Asunto(s)
Artemisininas/farmacología , Gosipol/farmacología , L-Lactato Deshidrogenasa/metabolismo , Praziquantel/farmacología , Schistosoma japonicum/enzimología , Animales , Arteméter , Catálisis/efectos de los fármacos , L-Lactato Deshidrogenasa/genética , Oxidación-Reducción/efectos de los fármacos , Proteínas Recombinantes/metabolismo , EspectrofotometríaRESUMEN
Embryonic stem cells can proliferate indefinitely and are capable of differentiating into derivatives of all three embryonic germ layers in vitro, including the neural lineage. The main objective of this study is to test the effects of neural stem cell conditioned medium on the neural differentiation of mouse embryonic stem cells. When cultured in neural stem cell conditioned medium, mouse embryonic stem cells can form floating cell spheres composed of many nestin-positive cells. After trypsinization and growth on gelatin, these embryonic stem cell-derived neural progenitor cells can be expanded for more than 3 months without loss of neural progenitor characteristics. Both neuronal and glial cells can be readily generated from these cells under differentiation conditions. Thus, neural stem cell conditioned medium is a highly potent reagent for inducing the development of mouse embryonic stem cells into the neural lineage, especially neural progenitor cells.