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Originally published In Press as doi:10.1074/jbc.M110275200 on February 1, 2002

J. Biol. Chem., Vol. 277, Issue 20, 17544-17547, May 17, 2002
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Selection and Identification of Dense Granule Antigen GRA3 by Toxoplasma gondii Whole Genome Phage Display*

Johan RobbenDagger , Kirsten HertveldtDagger §, Eugène Bosmans, and Guido VolckaertDagger ||

From the Dagger  Laboratory of Gene Technology, Katholieke Universiteit Leuven, Kasteelpark Arenberg 21, B-3001 Leuven, Belgium and  DiaMed Eurogen, Transportstraat 4, B-3980 Tessenderlo, Belgium

Toxoplasma gondii is a ubiquitous, unicellular, eukaryotic parasite with a complex intracellular life cycle capable of invading and chronically infecting a wide variety of vertebrate host species, including man. Although normally opportunistic in healthy adults, it is a lethal pathogen in immunocompromised humans, particularly in AIDS patients. We present the application of a genomic phage display as a tool for the direct identification of antigens with potential value in diagnosis and/or as subunit vaccine components. Using a polycosmid cloning strategy, we constructed a large phagemid display library (>109 independent clones) of mixed short genomic restriction fragments (<=  500 bp) of T. gondii genomic DNA (80 Mbp genome size) fused to gene III of the filamentous phage M13. Biopanning of the library with monoclonal Toxoplasma antibodies resulted in the isolation and identification of an epitope of GRA3, an antigen located in the dense granules of T. gondii tachyzoites. The reactivity of the phage displaying the GRA3 epitope with the monoclonal antibody was confirmed by an enzyme-linked immunosorbent assay. These results demonstrate the accessibility of midsized eukaryotic genomes to display technology and the feasibility to screen these whole genome display libraries with antibodies for isolating novel antigenic determinants.

* This work was supported by the Flemish Biotechnology Action Program (Vlaams Actieprogramma Biotechnologie, VLAB).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Recipient of a predoctoral fellowship from the Fonds voor Wetenschappelijk Onderzoek Vlaanderen (FWO).

|| To whom correspondence should be addressed. Tel.: 32-16-329669; Fax: 32-16-321965; E-mail: guido.volckaert@agr.kuleuven.ac.be.

Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
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