Subcloning and expression of the Brucella abortus L7/L12 ribosomal gene and T-lymphocyte recognition of the recombinant protein.

AUTOR(ES)

Oliveira, S C

RESUMO

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.

Documentos Relacionados

• Collaboration of bovine T lymphocytes and macrophages in T-lymphocyte response to Brucella abortus.
• Delayed-type hypersensitivity activity of the Brucella L7/L12 ribosomal protein depends on posttranslational modification.
• Ribosomal protein L7/L12 is required for optimal translation.
• Involvement of ribosomal protein L7/L12 in control of translational accuracy.
• Specificity of the H-2 L(d)-restricted cytotoxic T-lymphocyte response to the mouse hepatitis virus nucleocapsid protein.