Subcloning and expression of the Brucella abortus L7/L12 ribosomal gene and T-lymphocyte recognition of the recombinant protein.
The Brucella abortus L7/L12 ribosomal gene was amplified by PCR and subcloned into the prokaryotic expression vector pMAL-c2. Escherichia coli DH5 alpha was transformed with the pMAL-L7/L12 construct, and gene expression was induced by IPTG (isopropyl-beta-D-thiogalactopyranoside). The resulting fusion protein was purified by affinity chromatography and confirmed by Western blot (immunoblot) analysis using an anti-maltose-binding protein antibody. Additionally, purified recombinant L7/L12 protein induced T-lymphocyte proliferation of B. abortus-primed bovine peripheral blood mononuclear cells. Phenotypic analysis of the proliferating cell population demonstrated an increase in the percentage of CD4+ T lymphocytes when peripheral blood mononuclear cells were cultured with recombinant L7/L12 compared with cells cultured in medium alone. Subcloning and expression of a B. abortus gene encoding a previously demonstrated immunodominant protein for bovine lymphocytes are important steps in selecting Brucella proteins that have potential as a component of a genetically engineered candidate vaccine.
Oliveira
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SC
Department of Animal Health and Biomedical Sciences, University of Wisconsin-Madison 53706.
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D000818:Animals; D001483:Base Sequence; D002003:Brucella abortus / Q000235:genetics* / Q000276:immunology; D002417:Cattle; D003001:Cloning, Molecular; D017931:DNA Primers / Q000737:chemistry; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000276:immunology; D012269:Ribosomal Proteins / Q000235:genetics*; D013601:T-Lymphocytes / Q000276:immunology*
D017931:DNA Primers; D011994:Recombinant Proteins; D012269:Ribosomal Proteins; C016700:ribosomal protein L7-L12