Miranda-Miranda E, Murillo-Sánchez MH, Cossío-Bayúgar R

Idioma: Español
Referencias bibliográficas: 30
Paginas: 83-90
Archivo PDF: 193.03 Kb.

RESUMEN

Reportamos en este trabajo la clonación y expresión en el sistema de Baculovirus, de una cisteín proteasa (CP), del nematodo parásito gastroentérico de rumiantes Haemonchus contortus, con el objetivo de obtener con fines inmunodiagnósticos una proteasa recombinante con propiedades bioquímicas e inmunogénicas semejantes a las naturales aplicable a las campañas de diagnóstico, prevención y control de la hemoncosis ovina. Se aisló el gene correspondiente a la CP por medio de PCR, partiendo de cADN de H. contortus, la secuencia de ADN se clonó en fase con el promotor de polihedrina en un vector de expresión replicable en bacterias, se usaron células de lepidóptero Sf9 co-transfectadas simultáneamente con el CP-vector y ADN desnudo de baculovirus, las cuales originaron por recombinación homóloga, virus recombinantes con capacidad de expresar la CP en cultivos de células de insecto. La CP se identificó como un precursor inactivo fusionado con un sitio de afinidad 6XHis y se aisló por cromatografía de afinidad para generar CP activa enzimáticamente. La proteasa recombinante fue identificada por el suero de ovinos experimentalmente infestados con H. contortus tanto por inmunoelectrotransferencia como por ELISA, así como por su efecto catalítico sobre proteínas copolimerizadas en SDS-PAGE, el sistema de expresión en baculovirus mostró un rendimiento de 400 mg de antígeno diagnóstico recombinante por cada litro de medio (400 mg L^-1). Se concluye que la CP recombinante obtenida es semejante a la natural proveniente del parásito, se produce in vitro en grandes cantidades y puede ser aplicada en ensayos inmunodiagnósticos e inmunoprofilácticos destinados al inmunodiagnóstico y control de la hemoncosis de rumiantes.

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