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2013, Número 3

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Biotecnol Apl 2013; 30 (3)


Expresión y purificación de la proteína completa NS1 del virus dengue serotipo 2 a partir de un aislamiento

Lemos G, Guillén I, Fernández JR, Díaz T, Colarte AB, Fernández CME

Idioma: Ingles.
Referencias bibliográficas: 32
Paginas: 187-193
Archivo PDF: 206.27 Kb.


RESUMEN

El diagnóstico certero de la infección por el virus dengue (DENV) es esencial para su tratamiento oportuno. La proteína NS1 (46-50 kDa) es una glicoproteína del DENV de secuencia altamente conservada en los cuatro serotipos del virus, que se puede detectar durante la fase febril del dengue en pacientes infectados por primera o segunda vez, como marcador específico de la infección. Por ello, un test basado en la proteína NS1, pudiera facilitar su diagnóstico cuando se emplea junto con la detección de la inmunoglobulina M (IgM) contra el DENV. Entre las principales dificultades para su obtención está el aislamiento de la proteína NS1 de cultivos celulares de mamíferos, lo cual no es seguro, es laborioso y caro, y con bajos rendimientos que impiden su escalado. En este trabajo se clonó la secuencia completa de la proteína NS1 (rNS1) del DENV serotipo 2 en el vector pET28a, fusionado con una cola de histidina 6xHis en el extremo N-terminal. Esta proteína se obtuvo de forma recombinante en Escherichia coli, cepa Rosetta, como cuerpos de inclusión, con aproximadamente 46 kDa, y se purificó por cromatografía de afinidad de quelatos metálicos (IMAC) en condiciones desnaturalizantes. Sueros humanos de pacientes positivos al dengue mostraron reactividad contra la rNS1 en ensayos de ELISA y Western blot. La proteína rNS1 desnaturalizada se administró directamente como inmunógeno en ratones Balb/C, cuya respuesta de anticuerpos policlonales detectó a la proteína NS1 natural del DENV serotipo 1 en ensayos de inmunoblot.


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