RESUMO
A 28-kDa glutathione S-transferase (Cs28GST) was purified from a Clonorchis sinensis cytosolic fraction through anion-exchange and glutathione-affinity column chromatographies. A monoclonal antibody raised against Cs28GST reacted specifically to the C. sinensis antigen among trematode proteins. A putative peptide of 212 amino residues deduced from a cDNA clone appeared homologous with 28-kDa GST of trematodes, and its secondary structural elements predicted a GSH-binding site. Recombinant Cs28GST showed GST enzyme activity with CDNB substrate and was sensitive to the model inhibitors. The recombinant Cs28GST was antigenically indistinguishable from the native form and was recognized specifically by C. sinensis-infected human sera. The Cs28GST was localized in the tegument and underlying mesenchymal tissues. It is suggested that Cs28GST may play significant physiological roles against bioreactive molecules and be a useful reagent for serodiagnosis of clonorchiasis.
Assuntos
Clonorchis sinensis/enzimologia , Glutationa Transferase/genética , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Cromatografia de Afinidade , Cromatografia por Troca Iônica , Clonagem Molecular , Clonorchis sinensis/classificação , Clonorchis sinensis/genética , Análise por Conglomerados , DNA Complementar/química , Glutationa Transferase/química , Glutationa Transferase/isolamento & purificação , Humanos , Immunoblotting , Imuno-Histoquímica , Dados de Sequência Molecular , Filogenia , Reação em Cadeia da Polimerase , Coelhos , Proteínas Recombinantes/química , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Alinhamento de SequênciaRESUMO
In the course of immunoscreening of Clonorchis sinensis cDNA library, a cDNA CsRP12 containing a tandem repeat was isolated. The cDNA CsRP12 encodes two putative peptides of open reading frames (ORFs) 1 and 2 (CsRP12-1 and -2). The repetitive region is composed of 15 repeats of 10 amino acids. Of the two putative peptides, CsRP12-1 was proline-rich and found to have homologues in several organisms. Recombinant proteins of the putative peptides were bacterially produced and purified by an affinity chromatography. Recombinant CsRP12-1 protein was recognized by sera of clonorchiasis patients and experimental rabbits, but recombinant CsRP12-2 was not. One of the putative peptide, CsRP12-1, is designated CsPRA, proline-rich antigen of C. sinensis. Both the C-termini of CsRP12-1 and -2 were bacterially produced and analysed to show no antigenicity. Recombinant CsPRA protein showed high sensitivity and specificity. In experimental rabbits, IgG antibodies to CsPRA was produced between 4 and 8 weeks after the infection and decreased thereafter over one year. These results indicate that CsPRA is equivalent to a natural protein and a useful antigenic protein for serodiagnosis of human clonorchiasis.
