Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.7.6 (RNA polymerase)
 34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Two immunogenic proteins, Sm31 and Sm32, originating from the gut of Schistosoma mansoni were evaluated for their potential as recombinant immunodiagnostic reagents. Sm31 and Sm32 cDNA fragments were cloned and expressed in Escherichia coli as polypeptides fused to RNA polymerase of bacteriophage MS2. The recombinant proteins were tested in enzyme-linked immunosorbent assays (ELISA) with paired sera of 182 persons from Mali with S. mansoni and S. haematobium infections collected before and one year after treatment with praziquantel. Pretreatment sera of the study population gave a strong antibody response to Sm31 and Sm32 in immunoblots of total worm extract with the sera. The sensitivities of both western blotting (86%) and ELISA (75%) using Sm31 and Sm32 fusion proteins compared well with a single egg count (84%). Chemotherapy resulted in an overall decline of egg counts. Posttreatment sera gave significantly lower reactivities than the pretreatment sera. Our results demonstrate the feasibility of detecting circulating antibodies with recombinant antigens in schistosomiasis.
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PMID:Immunological analysis of cloned Schistosoma mansoni antigens Sm31 and Sm32 with sera of schistosomiasis patients. 179 25

We have expressed two cDNA sequences encoding 121 and 230 amino acids of the C-terminus of the Schistosoma mansoni Hsp 70 in Escherichia coli. The products were synthesized as polypeptides fused to the RNA polymerase of bacteriophage MS2, and their reactivities were tested in ELISAs, using sera from human and murine infections. Anti-Hsp70 antibodies were detected in a significant number of individuals suffering from chronic schistosomiasis mansoni, but not in patients with known recent infections. This, together with the finding that antibodies directed at S. mansoni-specific Hsp70 determinants during the course of infection of experimental mice were not detectable until 5-6 weeks post-infection, suggests that the protein may be a useful marker for distinguishing late and early infections. The diagnostic specificity of Hsp70 was evaluated with sera from humans infected with different schistosome species and other parasitic diseases. While some subjects infected with S. haematobium produced antibodies which recognized the S. mansoni Hsp70, no such antibodies were generated in S. japonicum infected individuals. However, cross-reactive antibodies were elicited in donors with other parasitic diseases such as filariasis and malaria. The absence of antibodies in early infection and the observed cross-reactivities led us to conclude that Hsp70 will be of limited value in the diagnosis of schistosomiasis.
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PMID:The humoral response to heat shock protein 70 in human and murine Schistosomiasis mansoni. 211 91

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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PMID:Cloning, molecular characterization of a 13-kDa antigen from Schistosoma japonicum, Sj13, a putative salivary diagnosis candidate for Schistosomiasis japonica. 1963 40

Schistosomes are the causative agent of schistosomiasis. The 70-kDa heat-shock proteins (HSP70) are considered the predominant HSP family and play a key regulatory role in parasite development and pathogenesis. Based on the published sequences in Genbank/EMBL, an open-reading frame (ORF) encoding 653 amino acids (XP_002581385.1) and belonging to the Schistosoma HSP70 protein family with a molecular weight of 71.49 kDa was identified by bioinformatic analysis. Since the sequence shared 77% identity with the published full-length Homo sapiens HSP70 protein, it was named Schistosoma mortalin-like protein (MLP/Hsp70). Here, we report the molecular and functional characterization of the Schistosoma japonicum SjMLP/hsp70 as a member of the HSP70 family. The complete SjMLP/hsp70 coding sequence was amplified from a S. japonicum adult worm cDNA library by polymerase chain reaction (PCR) and subcloned into the pET28a expression vector. The purified recombinant protein, rSjMLP/hsp70, was identified as a member of 70-kDa HSP family by mass spectrometry and could be recognized by the S. japonicum-infected mouse serum. Reverse transcriptase polymerase chain reaction (RT-PCR) and western blotting analysis revealed that SjMLP/hsp70 was widely expressed in the eggs, cercariae, schistosomula, and adult worms of S. japonicum. A thermotolerance assay showed that rSjMLP/hsp70 could protect Escherichia coli cells from heat damage. This chaperone-like activity was demonstrated by full-length SjMLP/hsp70. The detection of specific antibody levels by indirect enzyme-linked immunosorbent assay and IFN-gamma secretion of splenocytes by ELISpot assay suggested that mice immunized with SjMLP/hsp70 were able to elicit Th1-type bias immune response. The challenge-protective experiment showed that DNA vaccine of SjGST combined with SjMLP/hsp70 could induce a 31.31% reduction of worm burden and 58.59% reduction of egg burden in intestinal tissue of immunized mice. Our results imply that SjMLP/hsp70 has a potential adjuvant function and might be a vaccine candidate for schistosomiaisis, which is the first report of the expression and preliminary characterization analysis of this molecule.
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PMID:Molecular and functional characterization of a mortalin-like protein from Schistosoma japonicum (SjMLP/hsp70) as a member of the HSP70 family. 2060 14

Blood flukes or schistosomes are the causative agents of human schistosomiasis, one of the major neglected tropical diseases. Draft genome sequences have been reported for schistosomes, but functional genomics tools are needed to investigate the role and essentiality of the newly reported genes. Vector based RNA interference can contribute to functional genomics analysis for schistosomes. Using mRNA encoding reporter firefly luciferase as a model target, we compared the performance of a schistosome and a human promoter from the U6 gene in driving shRNA in human fibrosarcoma cells and in cultured schistosomes. Further, both a retroviral [Murine leukemia virus (MLV)] and plasmid (piggyBac, pXL-Bac II) vector were utilized. The schistosome U6 gene promoter was 270 bp in length, the human U6 gene promoter was 264 bp; they shared 41% identity. Following transduction of both HT1080 fibrosarcoma cells and schistosomules of Schistosoma mansoni with pseudotyped MLV virions, stronger knockdown of luciferase activity was seen with the virions encoding the human U6 promoter driven shRNA than the schistosome U6 promoter. A similar trend was seen after transfection of HT1080 cells and schistosomules with the pXL-Bac-II constructs-stronger knockdown of luciferase activity was seen with constructs encoding the human compared to schistosome U6 promoter. The findings indicate that a human U6 gene promoter drives stronger shRNA activity than its schistosome orthologue, not only in a human cancer cell line but also in larval schistosomes. This RNA polymerase III promoter represents a potentially valuable component for vector based RNA interference studies in schistosomes and related platyhelminth parasites.
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PMID:Human U6 promoter drives stronger shRNA activity than its schistosome orthologue in Schistosoma mansoni and human fibrosarcoma cells. 2195 24

Schistosoma japonicum is the pathogen responsible for schistosomiasis japonica, one of the major infectious diseases targeted for prevention nationally in China. Expression of heat shock proteins (HSPs) following stress plays a very important biological role in many organisms including S. japonicum. Among the HSP family, the 70-kDa HSPs are most responsible for intracellular chaperone and extracellular immunoregulatory functions. Based on the published sequences in GenBank/EMBL (AF044412.1), open reading frame belonging to HSP70 protein corresponds to a full-length cDNA containing an open reading frame of 1,947 bp encoded of 648 amino acids was identified as HSP70 from schistosome. In this study, the coding region that we named rSj648/hsp70 was amplified from S. japonicum adult worm cDNA library, and the recombinant protein was expressed in vector pET32a(+) and purified using a Ni-NTA purification system. The target protein rSj648/hsp70 was determined by matrix-assisted laser desorption/ionization mass spectrometer after thrombin digestion and dialysis. Reverse transcriptase polymerase chain reaction and Western blotting analysis confirmed that Sj648/hsp70 could be expressed in the eggs, normal cercariae, ultraviolet-attenuated cercariae (UVAC), and adult worms of S. japonicum. Real-time quantitative PCR analysis indicated that Sj648/hsp70 was expressed significantly higher in eggs than that in cercariae and adult worms, and the expression in UVAC was higher than that in normal cercariae. A thermotolerance assay showed that rSj648/hsp70 could protect Escherichia coli cells from heat damage. The detection of specific antibody levels by indirect enzyme-linked immunosorbent assay demonstrated that mice immunized with rSj648/hsp70 induced higher level of specific anti-rSj648/hsp70 IgG1 compared with those vaccinated with adjuvant alone, indicating that rSj648/hsp70 was able to elicit Th2-type bias immune response. Our results suggest that Sj648/hsp70 might be an important molecule in parasite-host interaction and display potential roles in mice immunoregulation system.
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PMID:Molecular cloning and characterization of a HSP70 gene from Schistosoma japonicum. 2207 51

Sj7TR is a 13 kDa repetitive region of a 31 kDa protein in Schistosoma japonicum known as Sjp_0110390 that showed high sensitivity and specificity in antibody detection against schistosomiasis patients. However, the current database for S. japonicum genes characterized it only as an expressed protein. A more thorough understanding of this antigenic protein is therefore necessary to possibly give more information about the nature of this protein and its role in the parasite. In this study, immunolocalization and expression profiling were done for Sjp_0110390 on the different stages of the parasite. Immunofluorescent assay showed that Sjp_0110390 was expressed in the young stages of the parasites including the schistosomula, eggs, aquatic and intra-molluscan stages. This was supported by the reverse-transcriptase PCR which confirmed the stage-specific expression of Sjp_0110390 and Western blot test which detected the protein in the extracted eggs proteins, but not in the adults. Furthermore, it was also highly expressed in infected Oncomelania hupensis nosophora snails suggesting that Sjp_0110390 might have a role in the development of the parasite inside the intermediate host. This result also suggests that Sj7TR might be used not only for human diagnosis but to detect snail infection as well.
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PMID:Localization and expression profiling of a 31 kDa antigenic repetitive protein Sjp_0110390 in Schistosoma japonicum life stages. 2325 1