A recombinant surface protein of Babesia bovis elicits bovine antibodies that react with live merozoites.
Ten monoclonal antibodies (MoAbs) were generated against five surface-exposed proteins (16 kDa, 42 kDa, 44 kDa, 60 kDa, 225 kDa) on merozoites of Babesia bovis. A genomic library constructed in the lambda gt11 expression vector was screened with MoAbs in a plaque immunoassay for identification of clones expressing recombinant surface proteins. Two recombinant clones were identified (lambda Bo44-15 and lambda Bo44-16) that encoded a protein recognized by a MoAb specific for an epitope on the native 44-kDa surface protein. Southern blot analysis using radiolabeled Bo44-15 DNA (1.25 kb) against merozoite DNA and bovine leukocyte DNA confirmed the parasite-specificity of the cloned insert and revealed multiple bands of hybridization with merozoite DNA. Western blot analyses of lambda Bo44-15 lysogen preparations demonstrated that recombinant protein production in this clone was IPTG-induced and that the recombinant molecule was a beta-galactosidase fusion protein. Additionally, recombinant 44-kDa protein, purified by immunoaffinity chromatography, reacted with specific MoAb in Western blot assay indicating that the integrity of the epitope was retained during purification. Immune sera from calves immunized with purified recombinant Bo44-15 protein immunoprecipitated metabolically radiolabeled merozoite protein of 44 kDa indicating that antibody induced by recombinant Bo44-15 protein recognized native 44-kDa protein. Also, these sera reacted with the surface of live merozoites as evidenced by indirect immunofluorescence assay. Serum antibody titers determined by this assay had a wide range.
Reduker
D W
DW
Department of Veterinary Microbiology and Pathology, Washington State University, Pullman.
Jasmer
D P
DP
Goff
W L
WL
Perryman
L E
LE
Davis
W C
WC
McGuire
T C
TC
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000913:Antibodies, Protozoan / Q000276:immunology*; D000953:Antigens, Protozoan / Q000276:immunology*; D000954:Antigens, Surface / Q000235:genetics / Q000276:immunology; D001403:Babesia / Q000235:genetics / Q000276:immunology*; D015139:Blotting, Southern; D015153:Blotting, Western; D002417:Cattle; D003001:Cloning, Molecular; D000939:Epitopes / Q000276:immunology; D009693:Nucleic Acid Hybridization; D011993:Recombinant Fusion Proteins / Q000276:immunology*; D011994:Recombinant Proteins / Q000276:immunology*
D000911:Antibodies, Monoclonal; D000913:Antibodies, Protozoan; D000953:Antigens, Protozoan; D000954:Antigens, Surface; D000939:Epitopes; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins