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- ItemSomente MetadadadosCharacterization of Schistosoma mansoni ATPDase2 gene, a novel apyrase family member(Elsevier B.V., 2007-01-12) Levano-Garcia, Julio; Mortara, Renato A.; Verjovski-Almeida, Sergio; DeMarco, Ricardo; Universidade de São Paulo (USP); Universidade Federal de São Paulo (UNIFESP)Schistosoma mansoni is a major causative agent of schistosomiasis, which constitutes a severe health problem in developing countries. We have previously described the SmATPDase1 gene, encoding a protein from the external surface of the parasites. in this work, we describe the cloning and characterization of SmATPDase2, a novel C1339-like ATP diphosphohydrolase gene in S. mansoni. in silico analysis of the protein encoded by SmATPDase2 predicts a single N-terminal transmembrane domain similar to that described for secreted human apyrase isoforms. Immuno-colocalization experiments detected both SmATPDase proteins at the S. mansoni adult worm tegument basal and apical membranes, but only SmATPDase2 in the tegument syncytium. SmATPDase2 but not SmATPDase1 protein was detected by Western blot in culture medium supernatants following incubation of adult worms in vitro, indicating that SmATPDase2 was secreted by the parasite to the medium. Taken together these data suggest a non-redundant role for SmATPDase2 in the parasite-host interplay. (c) 2006 Elsevier Inc. All rights reserved.
- ItemSomente MetadadadosCloning and characterization of a gene encoding a novel immunodominant antigen of Trypanosoma cruzi(Elsevier B.V., 1997-08-01) Lesenechal, M.; Duret, L.; Cano, M. I.; Mortara, R. A.; Jolivet, M.; Camargo, M. E.; daSilveira, J. F.; ParanhosBaccala, G.; ECOLE NORMALE SUPER LYON; LAB BIOMETRIE; Universidade Federal de São Paulo (UNIFESP); BIOLAB MERIEUXA Trypanosoma cruzi genomic expression library was screened with a pool of sera obtained from chronic chagasic patients. the recombinant antigen (Tc40) isolated from this library reacted with a large number of serum samples of chronic chagasic patients, suggesting that the presence of anti-Tc40 antibodies may be specifically associated to Chagas' disease. the full-length sequence of the Tc40 gene was determined after isolation of genomic and cDNA clones. the Tc40 cDNA includes a large open reading frame (2745 bp-long) that encodes a polypeptide of 100 kDa without any homology with previously described T. cruzi sequences. in contrast with other T. cruzi antigens whose immunodominant B-cell epitopes are composed by amino acid repetitive motifs, Tc40 does not show any amino acid repetition. Antibodies against the Tc40 recombinant protein reacted with three native polypeptides of 100, 41 and 38 kDa which are lightly associated with membranes or cytoskeleton and expressed in all developmental stages of the parasite life cycle. A transcript of 3.9-kb was detected in Northern blot analysis which is large enough to encode a 100 kDa polypeptide. Tc40 genes were mapped on a chromosomal band of 1.1 Mbp and in a few copies per haploid genome in the G strain. (C) 1997 Elsevier Science B.V.
- ItemSomente MetadadadosCloning and expression of a functionally active truncated N-glycosylated KSHVORF4/KCP/Kaposica in the methylotrophic yeast Pichia pastoris(New York Acad Sciences, 2005-01-01) Pereira, NAG; Juliano, M. A.; Carmona, A. K.; Sturrock, E. D.; Kotwal, G. J.; Lahiri, D. K.; Univ Cape Town; Universidade Federal de São Paulo (UNIFESP)Kaposi's sarcoma herpesvirus (KSHV) is a typical DNA virus that is associated with a number of proliferative diseases including Kaposi's sarcoma. the KSHV open reading frame (ORF) 4 encodes a complement regulatory protein (Kaposi complement control protein, KCP) that binds complement components and inhibits the complement-mediated lysis of cells infected by the virus, thus providing a strategy for evasion of the host complement system. Based on primary sequence analysis and comparison with other functionally and structurally similar proteins, oligonucleotide primers were designed to amplify by polymerase chain reaction (PCR) three regions of the predicted ORF 4 from human herpes virus-8 (HHV-8) DNA isolated from a primary effusion lymphoma cell line. the PCR products were inserted by ligation into the expression vector pPIC9 to generate three recombinant plasmids for heterologous expression in the yeast, Pichia pastoris, to produce separately the four N-terminal sushi domains (KCP-S, small), KCP protein lacking the putative transmembrane-binding domain (KCP-M, medium), and the full-length protein (KCP-F, full). Expression of the viral proteins was confirmed by SDSPAGE, immunologic detection, and Western blot analyses using a rabbit polyclonal antibody directed against a selected peptide region that is common to all three recombinant KCPs. KCP-S directly from expression media could inhibit complement-mediated lysis of sensitized sheep erythrocytes by approximately 60% in a hemolysis assay. This result confirms previous reports that recombinant KCP is twice as efficient in inhibiting the classic pathway-mediated lysis of erythrocytes than is the vaccinia virus complement control protein, which also contains four sushi domains.
- ItemSomente MetadadadosCloning, Characterization and Anti-Inflammatory Properties of Bothrops jararaca Snake Antithrombin(Bentham Science Publ Ltd, 2015-01-01) Morais-Zani, Karen de; Grego, Kathleen Fernandes; Torquato, Ricardo José Soares [UNIFESP]; Silva, Caroline Serino; Tanaka, Aparecida Sadae [UNIFESP]; Tanaka-Azevedo, Anita Mitico; Inst Butantan; Universidade de São Paulo (USP); Universidade Federal de São Paulo (UNIFESP)Antithrombin inhibits blood coagulation through the interaction with serine proteases in both intrinsic and extrinsic pathways. In addition, antithrombin also shows anti-inflammatory properties, which are independent of its effects on coagulation. This work shows for the first time the cloning and sequencing of antithrombin from a snake species. This predicted protein is composed by 430 amino acids and presents about 64.5% sequence identity to human antithrombin. Biacore experiments revealed that the binding affinity of Bothrops jararaca snake antithrombin to heparin was similar to 30 times higher than that of human antithrombin. Furthermore, Bothrops jararaca antithrombin is more effective in preventing acute inflammation induced by carrageenan when compared to human antithrombin. Hence, the results showed herein suggest that Bothrops jararaca antithrombin can play a key role in the control of acute inflammation and that this molecule might be used as a pharmacological tool and as a prototype for drug development.
- ItemSomente MetadadadosCloning, expression and characterization of Bauhinia variegata trypsin inhibitor BvTI(Walter de Gruyter & Co, 2005-11-01) Souza, A. F. de; Torquato, RJS; Tanaka, A. S.; Sampaio, CAM; Universidade Federal de São Paulo (UNIFESP)A Bauhinia variegata trypsin inhibitor (BvTI) cDNA fragment was cloned into the pCANTAB5E phagemid. the clone pAS 1.1.3 presented a cDNA fragment of 733 bp, including the coding region for a mature BvTI protein comprising 175 amino acid residues. the deduced amino acid sequence for BvTI confirmed it as a member of the Kunitz-type plant serine proteinase inhibitor family. the BvTI cDNA fragment encoding the mature form was cloned into the expression vector, pET-14b, and expressed in E coli BL2I (DE3) pLysS in an active form. in addition, a BvTI mutant form, r(mut)BvTI, with a Pro residue as the fifth amino acid in place of Leu, was produced. the recombinant proteins, rBvTI and r(mut)BvTI, were purified on a trypsin-Sepharose column, yielding 29 and 1.44 mg/I of active protein, respectively, and showed protein bands of approximately 21.5 kDa by SDS-PAGE. Trypsin inhibition activity was comparable for rBvTI (K-I=4 nm) and r(mut)BvTI (K-i=6 nm). Our data suggest that the Leu to Pro substitution at the fifth amino-terminal residue was not crucial for proteinase inhibition.
- ItemSomente MetadadadosMolecular characterization and immunolocalization of Schistosoma mansoni ATP-diphosphohydrolase(Elsevier B.V., 2003-08-08) DeMarco, R.; Kowaltowski, A. T.; Mortara, R. A.; Verjovski-Almeida, S.; Universidade de São Paulo (USP); Universidade Federal de São Paulo (UNIFESP)Schistosoma mansoni, a human parasite that constitutes a major health problem in developing countries, escapes from host defenses and survives in the human bloodstream. Here, we report the cloning of a S. mansoni ATP-diphosphohydrolase ortholog (SmATPDase1). Southern blots indicated that in S. mansoni it is a single-copy gene. RT-PCR revealed that SmATPDase1 is expressed in five stages of the parasite life cycle, namely cercaria, schistosomula, adults, eggs, and miracidia. Using confocal microscopy, SmATPDase1 protein was immunolocalized on the external surface in all stages, except eggs, being conspicuously present in adults. ATPDase, which is present on the outer surface of endothelial cells lining human blood vessels, has been implicated in thromboregulation by promoting ADP hydrolysis and inhibition of platelet aggregation. the presence of an ATPDase ortholog on the surface of S. mansoni suggests that the enzyme might play a role in the escape from host defenses that would involve platelet activation. (C) 2003 Elsevier Inc. All rights reserved.