Oliveira S C, Splitter G A
Department of Animal Health and Biomedical Sciences, University of Wisconsin-Madison 53706.
Infect Immun. 1994 Nov;62(11):5201-4. doi: 10.1128/iai.62.11.5201-5204.1994.
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.
通过聚合酶链反应（PCR）扩增流产布鲁氏菌L7/L12核糖体基因，并将其亚克隆到原核表达载体pMAL-c2中。用pMAL-L7/L12构建体转化大肠杆菌DH5α，并用异丙基-β-D-硫代半乳糖苷（IPTG）诱导基因表达。通过亲和层析法纯化所得融合蛋白，并用抗麦芽糖结合蛋白抗体通过蛋白质免疫印迹（免疫印迹）分析进行确认。此外，纯化的重组L7/L12蛋白可诱导经流产布鲁氏菌致敏的牛外周血单个核细胞的T淋巴细胞增殖。当外周血单个核细胞与重组L7/L12一起培养时，与仅在培养基中培养的细胞相比，增殖细胞群体的表型分析表明CD4 + T淋巴细胞的百分比增加。对一种编码先前已证明对牛淋巴细胞具有免疫显性的蛋白的流产布鲁氏菌基因进行亚克隆和表达，是选择具有作为基因工程候选疫苗成分潜力的布鲁氏菌蛋白的重要步骤。
