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Revista Colombiana de Química

versão impressa ISSN 0120-2804

Resumo

MORENO-GONZALEZ, Paula A; DIAZ, Gonzalo J  e  RAMIREZ-HERNANDEZ, María H. Production and purification of avian antibodies (IgYs) from inclusion bodies of a recombinant protein central in NAD+ metabolisim. Rev.Colomb.Quim. [online]. 2013, vol.42, n.2, pp.12-20. ISSN 0120-2804.

In contrast with other animal models, the use of hens for polyclonal antibodies production not only reduces animal intervention, but also increases the quantity of the obtained immunoglobulins. The phylogenetic distance between birds and mammals, 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 leads to the formation of insoluble and useless aggregates (inclusion bodies, IC). This article presents a strategy to produce avian polyclonal antibodies (IgYs) from IC. In order to obtain the antigen, the Giardia intestinalis nicotinamide mononucleotide adenylyltransferase recombinant protein (His-GiNMNAT) was expressed in Escherichia coli. The His-GiNMNAT protein was purified through IC solubilization and re-folding. The purified protein was use to immunize hens. The antibodies were purified from egg yolk by water dilution, followed by ammonium sulfate precipitation and thiophilic affinity chromatography. Specificity of the purified antibodies was tested against the His-GiNMNAT protein through Western blot essays. In terms of yield, 14.4 mg of highly pure IgYs were obtained from one egg yolk; these antibodies were able to detect 15 ng of antigen. IgYs specificity was improved by means of antigen affinity purification, allowing its implementation for endogenous detection of GiNMNAT protein in G. intestinalis.

Palavras-chave : Antibodies; IgYs; Inclusion bodies; Giardia intestinalis; NAD+; NMNAT.

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