Construction of a Babesia bovis expression library : isolation and characterization of clones encoding Babesia proteins

Abstract

Babesia bovis is a tick-transmitted, intraerythrocytic protozoan parasite causing a hemolytic disease in cattle throughout the world. To identify and characterize gene products produced during the parasite life cycle, a lambda gt11 expression library was constructed using genomic DNA prepared from in vitro erythrocyte cultures of B. bovis merozoites and digested with mung bean nuclease. This enzyme has been shown to cleave both genomic and cloned DNA at positions flanking the 5' and 3' ends of gene coding sequences but not within the genes. Recombinant bacteriophage that contain relevant translation products were identified by immunoscreening with a panel of polyclonal antisera which had been characterized by indirect immunofluorescent antibody assay and immunoblot analysis for specific antibody against B. bovis antigens. Multiple recombinants were detected and three were isolated for analysis and characterization. Fusion proteins produced from the B. bovis recombinants were used to affinity purify clone-specific immunoglobulins from polyvalent anti-B. bovis immune sera. These antibodies, in turn, were used in an immunofluorescence assay to verify the stage specific nature and the cellular location of proteins encoded by the recombinants. The clone-specific antibodies were also utilized in an immunoblot assay to determine the molecular weight of the native Babesia proteins encoded by the cloned genes and to test for cross reactivity of the recombinant proteins with host antigens and protein preparations of other Babesia species. The insert size was determined by restriction mapping of the recombinant phage and the cloned DNA was examined for parasite and species specificity by Southern blot analysis.