Análisis estequiométrico de la reacción de conjugación del péptido P0 a la proteína BM86 empleando el conector MPS
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Fecha
2016
Autores
Pérez Gómez, Lianny
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Universidad Central “Marta Abreu” de Las Villas. Facultad de Química Farmacia. Departamento de Licenciatura en Química
Resumen
Una de las nuevas estrategias en el campo de la medicina constituyen los preparados vacunales basados en péptidos sintéticos. Actualmente en el CIGB de Camagüey se está trabajando en la elaboración de un vacuna contra enfermedades hemoparasitarias en perros cuyo principio activo es el péptido sintético P0. Para aumentar su inmunogenidad y evitar que se degrade en el organismo del mamífero, el péptido P0 se conjuga a la proteína BM86 empleando el conector cruzado heterobifuncional MPS. El objetivo de este trabajo fue determinar la relación molar en que deben ser mezclados la proteína BM86, el péptido P0 y el reactivo MPS durante la reacción de conjugación, de manera que no se genere un exceso de ninguno de estos reactivos y tratando de conjugar la mayor cantidad de péptido posible para lograr aumentar su inmunogenidad. A esta reacción de conjugación se le dio seguimiento mediante el ensayo de Ellman para la determinación de grupos sulfihidrilos libres. Se obtuvo que como promedio por cada mol de proteína BM86 se logran conjugar 47 moles de MPS y 45 moles de P0. Paralelamente se demostró que el método de Ellman es una herramienta factible para el seguimiento de la reacción de conjugación y se determinó que disminuyendo la concentración de oxígeno disuelto en el medio de reacción se logra la mayor estabilidad del péptido P0 a la dimerización.
Synthetic peptides are one of the new strategies in the field of medicine to vaccine formulation. Currently in the CIGB in Camagüey it is working on developing a vaccine against blood parasitic diseases in dogs. The active ingredient of this vaccine is the synthetic peptide P0. To increase its immunogenicity and avoid degradation in the mammalian body, the P0 peptide is conjugated to the protein BM86 using heterobifunctional cross linker MPS. The aim of this study was to determine the molar ratio to be mixed the BM86 protein, P0 peptide and the MPS reagent during the conjugation reaction, so that an excess of any of these reagents is not generated and trying to combine peptide as much as possible to achieve increase its immunogenicity. The conjugation reaction was followed by Ellman's test for free sulfhydryl determination groups. Was obtained that 47 moles of MPS were conjugated as an average per mole of protein BM86 and 45 moles of peptide P0. Ellman’s method was useful as a tool to follow the conjugation reaction. Lowering the dissolved oxygen concentration in the reaction media can achieve higher P0 peptide stability to dimerization.
Synthetic peptides are one of the new strategies in the field of medicine to vaccine formulation. Currently in the CIGB in Camagüey it is working on developing a vaccine against blood parasitic diseases in dogs. The active ingredient of this vaccine is the synthetic peptide P0. To increase its immunogenicity and avoid degradation in the mammalian body, the P0 peptide is conjugated to the protein BM86 using heterobifunctional cross linker MPS. The aim of this study was to determine the molar ratio to be mixed the BM86 protein, P0 peptide and the MPS reagent during the conjugation reaction, so that an excess of any of these reagents is not generated and trying to combine peptide as much as possible to achieve increase its immunogenicity. The conjugation reaction was followed by Ellman's test for free sulfhydryl determination groups. Was obtained that 47 moles of MPS were conjugated as an average per mole of protein BM86 and 45 moles of peptide P0. Ellman’s method was useful as a tool to follow the conjugation reaction. Lowering the dissolved oxygen concentration in the reaction media can achieve higher P0 peptide stability to dimerization.
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Palabras clave
Medicina Veterinaria, Proteínas Portadoras, Proteína Portadora BM86, Vacunas Peptídicas, Garrapata Marrón del Perro, Rhipicephalus sanguineus