Molecular cloning, characterization, and immunogenicity of a species-specific Babesia microti antigen.

摘要

The laboratory strain of the intraerythrocytic protozoan parasite, Babesia microti, produces an acute, lethal infection in mice. This species also causes a malaria-like disease in humans.;B. microti genomic DNA was prepared from parasitized murine erythrocytes, digested with mung bean nuclease to generate mostly intact gene fragments, and cloned into the lambda gt11 expression vector. Polyspecific antisera obtained from either chronically or acutely infected mice or from guinea-pigs immunized with solubilized B. microti antigens were used to screen the genomic expression library. These sera were previously characterized for parasite reactivity using immunoblot and indirect fluorescence assays (IFA). A recombinant phage, designated Bm13, was identified using the hyperimmune murine antisera, isolated and characterized. Clone Bm13 is 3.3 Kb in size. Southern hybridization analysis, in which both restriction fragments of B. microti and murine leukocyte genomic DNA were probed with radioactively labelled Bm13 (insert) DNA, confirmed the parasite specificity of the clone. In addition, the species specificity of clone Bm13 was also established since only genomic DNA isolated from the acute, virulent strain of B. microti contained any complementary sequences which hybridized to Bm13. Genomic DNA prepared from a chronic, attenuated strain of B. microti or from B. bovis did not contain any complementary sequences. The fusion protein encoded by Bm13 was used to affinity-purify monospecific antibodies from the polyspecific screening sera. Clone-selected antibodies recognized a native babesial polypeptide of 54kD.;Using a commercially prepared immunoaffinity adsorbent, the LacZ fusion protein, as well as non-fused Beta-galactosidase, was purified from crude recombinant and non-recombinant lysogen extracts, respectively. The affinity-purified fusion protein was characterized with respect to size and determined to be approximately 153kD.;Female BALB/C mice were immunized with fusion or Beta-galactosidase proteins to evaluate the potential immunogenicity of the fused polypeptide. Subsequently, the mice were intraperitoneally challenged with virulent B. microti merozoites. All mice, regardless of treatment, succumbed to the lethal challenge of B. microti-infected erythrocytes. Western blot analysis indicated that all immunized mice responded by producing peptide-specific antibodies. However, the antibody response was directed to the Beta-galactosidase portion rather than the babesia encoded region of the fusion protein.