RESUMEN
Researches on genes expressed in a cercarial stage-specific manner may help us understand the molecular events and functions during schistosome invasion of skin. A genomic clone encoding 8-kDa calcium-binding protein (SjCa8) specifically expressed in cercariae and skin-stage schistosomulum (transformed within 3 h) was obtained from cercariae. Recombinant protein was expressed in vector pET32a (+) and purified using a Ni-NTA purification system. The target protein SjCa8 was determined by matrix-assisted laser desorption/ionization time-of-flight (TOF)/TOF mass spectrometer after thrombin digestion and dialysis. Reverse transcriptase polymerase chain reaction and Western blot revealed SjCa8 can be detected in cercaria and skin-stage schistosomulum but not lung-stage schistosomulum, adult, or egg and was localized to head gland, penetration gland tubes, and penetration glands where Ca(2+) was abundant, and the cercarial tegument (but not tegument of tail) and body-tail junction. Furthermore, SjCa8 was interestingly detected in cercarial secretions. The characterization of SjCa8 indicated that it may undergo structural and physiological modifications, including repair of the surface membrane, changes in permeability that account for the loss of water tolerance, activities of calcium-depending enzymes, and immune signaling, etc. Furthermore, vaccination with rSjCa8 plus adjuvant induced protective effect with 50.39% worm reduction rate and significantly high hepatic and intestine egg reduction rates (54.16%, 50.63%, respectively), which is possibly mediated through an apparent induction of Th1-type immune response for strikingly high level of IgG2a and IgG2b developed in immunized C57BL/6 mice.
Asunto(s)
Antígenos Helmínticos , Proteínas de Unión al Calcio , Schistosoma japonicum/inmunología , Esquistosomiasis Japónica , Vacunas Sintéticas , Animales , Anticuerpos Antihelmínticos/sangre , Antígenos Helmínticos/genética , Antígenos Helmínticos/inmunología , Antígenos Helmínticos/metabolismo , Western Blotting , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/inmunología , Proteínas de Unión al Calcio/metabolismo , Proteínas del Helminto/genética , Proteínas del Helminto/inmunología , Proteínas del Helminto/metabolismo , Inmunización , Inmunohistoquímica , Estadios del Ciclo de Vida , Ratones , Ratones Endogámicos C57BL , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Schistosoma japonicum/genética , Schistosoma japonicum/crecimiento & desarrollo , Schistosoma japonicum/metabolismo , Esquistosomiasis Japónica/inmunología , Esquistosomiasis Japónica/parasitología , Esquistosomiasis Japónica/prevención & control , Células TH1/inmunología , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunologíaRESUMEN
OBJECTIVE: To analyze the difference of GRA7 gene of Toxoplasma gondii different isolated strains and express GRA7 in Escherichia coli. METHODS: The GRA7 gene was amplified from genomes of T. gondii isolates by PCR and was cloned into pGEX-4T-1. The recombinant plasmid was transformed into JM109 and sent to be sequenced. The sequence was analyzed with CLUSTALW (an internet tool). The recombinant plasmid was induced by IPTG to express the fusion protein,which was identified by SDS-PAGE and Western blot with positive sera. The protein was purified and used as a diagnostic antigen for ELISA to test serum samples. RESULTS: There was no difference among the sequences of T. gondii GRA7 gene from different isolates. The recombinant plasmid pGEX-4T-1/GRA7 induced by IPTG was expressed in E. coli. It was a GST fusion protein and could react with human and rabbit positive sera analyzed by Western blot. CONCLUSION: The GRA7 gene of T. gondii isolates is highly conservative. The GRA7 is expressed as a recombinant protein in Escherichia coli, which shows an immunoreactivity.