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目的了解羊种布鲁氏菌Omp19基因在真核细胞中的表达和对细胞免疫的影响。方法根据GenBank公布的羊布鲁氏菌16M株外膜蛋白Omp19基因序列设计1对引物,以其全基因组为模板进行扩增,Omp19基因扩增片段连接pMD18-T载体,转染及测序鉴定正确后进行双酶切,构建重组质粒pLEX-MSC-Omp19,转染巨噬细胞RAW264.7中,用Western blot分析Omp19基因在巨噬细胞内的表达,酶联免疫吸附试验(ELISA)检测转染Omp19基因的细胞干扰素-γ(IFN-γ)和IL-6分泌的影响。结果获得534bp目的基因片段并成功构建Omp19真核表达载体,Omp19基因在巨噬细胞内成功表达,表达蛋白Omp19可显著提高细胞分泌IFN-γ和IL-6水平(P<0.01)。结论 Omp19基因表达产物在细胞内具有免疫原性,能提高细胞免疫水平,可作为亚单位疫苗候选基因。
Objective To understand the expression of Brucella Omp19 gene in eukaryotic cells and its effect on cellular immunity. Methods A pair of primers was designed according to the Omp19 gene sequence of 16M Outer Membrane Protein (OMP19) of Brucella abortus from GenBank. The full-length genomic DNA was used as a template to amplify the Omp19 gene. The amplified fragment of Omp19 gene was ligated into pMD18-T vector and identified by transfection and sequencing After double enzyme digestion, the recombinant plasmid pLEX-MSC-Omp19 was constructed and transfected into RAW264.7 macrophages. The expression of Omp19 gene in macrophages was detected by Western blot. The expression of Omp19 gene in macrophages was detected by enzyme-linked immunosorbent assay (ELISA) Effect of Omp19 Gene on Interferon-γ (IFN-γ) and IL-6 Secretion. Results The target gene fragment of 534bp was obtained and the Omp19 eukaryotic expression vector was successfully constructed. Omp19 gene was successfully expressed in macrophages. The expression of Omp19 protein significantly increased the levels of IFN-γ and IL-6 secreted by cells (P <0.01). Conclusion The Omp19 gene expression product has immunogenicity in the cell, can improve the cellular immunity level, and can be used as subunit vaccine candidate gene.