RESUMEN
Sm13, a 13-kDa Schistosoma mansoni tegumental antigen, is one of the principal polypeptides recognized by antibodies from mice protectively vaccinated with adult-worm tegumental membranes. To obtain the complete gene encoding Sm13 we subcloned and sequenced a cDNA and a fragment of a genomic clone. The collated sequence contains 1,088 nucleotides and represents the full-length open reading frame of the gene, encoding a protein of 104 amino acids with a calculated molecular mass of 11,923 Da, compatible with the native protein identified in the tegumental membranes. The sequence derived from genomic DNA contains a 45-nucleotide intron. The analysis of the predicted protein suggests the presence of both N- and C-terminal hydrophobic membrane-spanning segments, and the coding region contains no homology in the currently available data bases. Additionally, the coding region is preceded by putative CCAAT and TATA boxes that may be involved in the control of expression. Western-blot analysis and indirect immunofluorescence resulted in the identification of a 13-kDa protein (Sm13) in the tegument of adult worms. The present study reveals that Sm13 behaves as an integral membrane protein upon partitioning in Triton X-1 14 and that it is present in worms of 3 weeks or older but not in schistosomula or miracidia. Moreover, it is also specifically recognized by sera from some schistosomiasis patients in enzyme-linked immunosorbent assay and Western-blot analysis, suggesting that it is immunogenic in human schistosomiasis.
Asunto(s)
Antígenos Helmínticos/genética , Antígenos de Superficie/genética , ADN de Helmintos/análisis , Proteínas del Helminto , Schistosoma mansoni/inmunología , Secuencia de Aminoácidos , Animales , Antígenos Helmínticos/análisis , Antígenos de Superficie/análisis , Secuencia de Bases , Southern Blotting , Western Blotting , Membrana Celular/química , Membrana Celular/inmunología , Clonación Molecular , Ensayo de Inmunoadsorción Enzimática , Inmunohistoquímica , Proteínas de la Membrana/análisis , Proteínas de la Membrana/genética , Proteínas de la Membrana/inmunología , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , ARN de Helminto/análisis , Schistosoma mansoni/química , Schistosoma mansoni/genéticaAsunto(s)
Antígenos Helmínticos/análisis , Interacciones Huésped-Parásitos/genética , Interacciones Huésped-Parásitos/inmunología , Schistosoma mansoni/genética , Schistosoma mansoni/inmunología , Animales , Antígenos de Superficie/genética , Antígenos de Superficie/inmunología , ADN Complementario/análisis , ADN Complementario/genéticaRESUMEN
Sm15 and Sm13 are recognized by antibodies from mice protectively vaccinated with tegumental membranes, suggesting a potential role in protective immunity. In order to raise antibodies for immunochemical investigations, the genes for these antigens were expressed in pGEX and pMal vectors so that comparisons could be made among different expression systems and different genes. The fusion proteins corresponding to several parts of the gene for the precursor of Sm15 failed in producing antibodies recognizing the parasite counterpart. On the other hand, antibodies raised against Sm13 MBP-fusion proteins recognized the 13 kDa tegumental protein. Thus the peculiarities of the gene of interest are important and the choice of the expression system must sometimes be decided on an empirical basis.