Evaluación preliminar de un sistema biológico eucariota (Nicotiana tabacum) escalable para la producción de insulina humana recombinante
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La diabetes mellitus es un problema de salud mundial cuyo tratamiento representa presión económica para los pacientes y los sistemas de salud pública. La producción de insulina, principal tratamiento de esta enfermedad es costosa y por ende se requieren métodos alternativos y económicos para aumentar su disponibilidad y efectividad para los pacientes. En este trabajo se emplearon plantas de tabaco (Nicotiana tabacum) como modelo biotecnológico para introducir genéticamente los genes requeridos para sintetizar y liberar insulina humana. Para ello, la secuencia codificante se clonó en casetes de expresión nuclear y plastidial para material vegetal y su correcta inserción se verificó mediante análisis de PCR del ADN genómico de los clones derivados. Además, se cuantificó la expresión y liberación de proteínas recombinantes mediante ELISA. La concentración alcanzó niveles promedio de 800pmol/L en los sobrenadantes de los derivados del constructo pAF_ins, lo que confirma la efectividad de la transformación plastidial. Para probar preliminarmente la bioactividad de la hormona, se evaluó su capacidad para inducir la fosforilación de su receptor en células HepG2. Los resultados mostraron un aumento en la fosforilación después de la estimulación tanto con insulina recombinante disponible comercialmente (control positivo), como con la molécula recombinante secretada por las plantas de tabaco. No se observó fosforilación al estimular las células con el medio de inanición (control negativo) o extractos de plantas silvestres. Los resultados muestran que N. tabacum es una plataforma viable y apropiada para la producción de insulina humana recombinante en su configuración funcional para aplicaciones terapéuticas.
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