RESUMEN
Sj20.8 gene was amplified by PCR and inserted into eukaryotic expression plasmid pcDNA3.1 to construct recombinant plasmid pcDNA3.1/Sj20.8, which was then injected into the quadriceps femoris of the BALB/c mice. Results showed that the Sj20.8 antigen was low expressed in the local tissue of the mice, and was not able to significantly reduce eggs in the liver than in the control mice.
Asunto(s)
Proteínas del Helminto/genética , Schistosoma japonicum/inmunología , Esquistosomiasis Japónica/inmunología , Vacunas de ADN/inmunología , Animales , ADN Recombinante/inmunología , Femenino , Biblioteca de Genes , Inmunización , Hígado/efectos de los fármacos , Hígado/parasitología , Ratones , Ratones Endogámicos BALB C , Recuento de Huevos de Parásitos , Plásmidos/genética , Distribución Aleatoria , Schistosoma japonicum/genética , Esquistosomiasis Japónica/parasitología , Esquistosomiasis Japónica/prevención & control , Vacunas de ADN/administración & dosificación , Vacunas de ADN/genéticaRESUMEN
OBJECTIVE: To acquire and analyze adult stage Schistosoma japonicum (Chinese strain) expressed sequence tags and new genes from an adult S. japonicum cDNA library, and to search new vaccine candidates and drug targets. METHODS: A cDNA library was constructed from adult stage S. japonicum. Clones were selected randomly from the cDNA library and were sequenced. ESTs and new genes were acquired after analysis in GenBank databases by BLAST and other programs. All ESTs and new genes were submitted to GenBank and received accession numbers. RESULTS: 149 ESTs were acquired from a total 382 clones that were randomly selected from the adult S. japonicum cDNA library. All ESTs were successfully submitted to the dbEST at Genbank. Some of them were homologous with sequences of male, female, egg, schistosomula, cercaria and miracidia of S. japonicum. 18 new genes of adult S. japonicum were acquired. Some genes were housekeeping genes and some genes might be interesting as vaccine candidates or drugs targets. CONCLUSIONS: The EST strategy is a rapid, efficient and economical method to acquire ESTs and to discover new genes of adult stage S. japonicum from cDNA libraries.
Asunto(s)
Etiquetas de Secuencia Expresada , Genes de Helminto , Schistosoma japonicum/genética , Animales , Clonación Molecular , ADN Complementario/química , Biblioteca de GenesRESUMEN
OBJECTIVE: To construct a recombinant prokaryotic expression vector (plasmid) containing microneme protein 1 (MIC1) partial gene in toxoplasma gondii (T. gondii) ZS2 isolate. The gene was expressed in varied Escherichia coli (E. coli) after sequencing. METHODS: The gene fragment coding MIC 1 from the genomic DNA of T. gondii ZS2 isolate was amplified by polymerase chain reaction (PCR). The gene was inserted to a prokaryotic expression vector pWR450-1 by digesting with restriction enzymes and linking reaction. The positive clone was screened on LB plates containing ampicillin and identified by restrictive enzyme digestion, PCR amplification and sequence analysis. The recombinant plasmid was transferred into E. coli TG1, JM109 (DE3) and DH5 alpha, and was expressed under the induction of IPTG. The expression products were identified by SDS-PAGE. The MIC1 gene structure was analyzed and compared in homology with the gene sequence of RH isolate using computer software. RESULTS: The recombinant plasmid pWR450-1/MIC1, after cloning from acquired 471 bp MIC1 gene fragment and amplified from the genome gene ZS2, was complete homologous to the sequence of RH isolate, reflecting its highly conservative. The gene could be expressed as fusion protein with 70,000 in varied E. coli. CONCLUSION: Recombinant plasmid pWR450-MIC1 was successfully constructed and could be expressed in different strains of E. coli, laying a foundation for research on its structure and function.