Producción y purificación de anticuerpos aviares (IgYs) a partir de cuerpos de inclusión de una proteína recombinante central en el metabolismo del NAD+
Production and purification of avian antibodies (IgYs) from inclusion bodies of a recombinant protein central in NAD+ metabolism
Keywords:
Anticuerpos, IgYs, Cuerpos de Inclusión (CI), Giardia intestinalis, NAD , NMNAT (es)Antibodies, IgYs, Inclusion bodies, Giardia intestinalis, NAD , NMNAT (en)
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El uso de gallinas para la producción de anticuerpos policlonales, reduce la intervención animal, obteniendo además una gran cantidad de anticuerpos. Las aves presentan mayor distancia filogenética entre sus antígenos y los de mamíferos ofreciendo altos porcentajes de anticuerpos específicos. La producción de anticuerpos requiere antígenos en cantidades considerables, por ello es creciente el uso de proteínas recombinantes para tales fines. No obstante, la obtención de proteínas en sistemas heterólogos conduce frecuentemente a la precipitación en agregados insolubles de limitada utilidad (cuerpos de inclusión).
Este trabajo presenta una metodología para la producción de anticuerpos policlonales (IgYs) empleando cuerpos de inclusión (CI) como antígeno. Los CI purificados e inoculados corresponden a la Nicotinamida / Nicotinato Mononucleotido Adenililtranferasa (His-GiNMNAT) de Giardia intestinalis expresada en Escherichia coli. La purificación del antígeno se llevó a cabo mediante solubilización y renaturalización. Los anticuerpos se purificaron de la yema de huevo de gallinas imunizadas mediante dilución en agua, seguida de precipitación con sulfato de amonio al 60% y afinidad tiofilica. Los anticuerpos fueron evaluados mediante inmunoblot empleando la proteína His-GiNMNAT. De una yema de huevo se obtuvieron 14,4 mg de IgYs, con alta pureza y con un reconocimiento de hasta 15ng de His-GiNMNAT. Se mejoró la especificidad de los IgYs mediante una purificación adicional por afinidad al antígeno, lo cual permitiría su empleo para el reconocimiento de la proteína del parásito.
The use of hens for the production of polyclonal antibodies reduces animal intervention and moreover yields a higher quantity of antibodies than other animal models. The phylogenetic distance between bird and mammal antigens, often leads to more specific avian antibodies than their mammalian counterparts.
Since a large amount of antigen is required for avian antibody production, the use of recombinant proteins for this procedure has been growing faster over the last years. Nevertheless, recombinant protein production through heterologous systems frequently prompts the protein to precipitate, forming insoluble aggregates of limited utility (inclusion bodies). A methodology for the production of avian polyclonal antibodies, using recombinant protein from inclusion bodies is presented in this article.
In order to produce the antigen, a recombinant Nicotinamide mononucleotide adenylyltransferase from Giardia intestinalis (His-GiNMNAT) was expressed in Escherichia coli. The protein was purified through solubilization from inclusion bodies prior to its renaturalization. Antibodies were purified from egg yolk of immunized hens by water dilution, followed by ammonium sulfate precipitation and thiophilic affinity chromatography.
The purified antibodies were tested against His-GiNMNAT protein in Western blot essays. From one egg yolk, 14.4 mg of highly pure IgY were obtained; this antibody was able to detect 15ng of His-GiNMNAT. IgY specificity was improved by means of antigen affinity purification, allowing its use for parasite protein recognition.
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