Omp25蛋白的真核表达及其对巨噬细胞的作用

本试验旨在构建表达猪布鲁菌（Brucellasuis）外膜蛋白0mp25蛋白的真核重组表达质粒，并探讨Omp25蛋白对猪肺泡巨噬细胞生长活性及细胞因子分泌的影响。以猪布鲁菌基因组DNA为模板，设计带有6×His标签的引物克隆Orap25基因，扩增产物克隆入pCI-neo真核表达载体，双酶切及测序鉴定正确后转染猪肺泡巨噬细胞3D4／21，检测其对巨噬细胞生长活性和分泌细胞因子的影响。RT—PCR和Westernblotting检测结果证实Omp25蛋白获得正确表达。显微镜观察细胞形态表明，Omp25蛋白对猪肺泡巨噬细胞的形态无明显影响。MTT结果表明，Orap25蛋白对猪肺泡巨噬细胞的生长活性有轻度但不显著的抑制作用。ELISA检测TNF—α和IL-12蛋白表达水平表明，Omp25蛋白可以显著抑制巨噬细胞分泌TNF-α和IL-12。本试验成功构建了表达猪布鲁菌外膜蛋白Omp25蛋白的真核重组表达质粒，该蛋白对猪肺泡巨噬细胞的细胞形态没有明显影响，对其生长活性有轻度但不显著的抑制作用，可以显著抑制巨噬细胞分泌TNF—α和IL-12。

Construction of Omp25 eukaryotic expression vector and analysis of its effects on porcine macrophage

In order to analyze the function of Omp25 protein and its role in Brucella escaping from the immune system,we constructed the eukaryotic expression vector pCI-neo-omp25 and studied its effects on cellular growth activity and the secretion of cytokine in 3D4/21 cells. Using DNA extracted from Brucella suis as a template,the gene of omp25 was amplified by PCR and then inserted into a eukaryote vector pCI-neo. The recombinant protein Omp25 was analyzed by RT- PCR, Western blotting, microscopy, MTT and ELISA. After transfection, Omp25 protein was expressed. The results of microscopy showed that Omp25 protein had no significant effect on cell morphology. MTT results indicated that Omp25 protein had not significantly inhibitory effect on the growth activity of 3D4/21 cells. ELISA results showed that Omp25 protein suppressed the expression of the TNF- and IL-12. The recombinant plasmid that expressed Omp25 protein was successfully constructed. The present results indicated that Omp25 protein suppressed the production of TNF-α and IL-12 secreted by macrophage and played an important role in immunosuppression of Brucella.