布氏杆菌BCSP_(31)/pVAX1重组表达质粒的构建及其免疫保护效果的研究

目的构建布氏杆菌BCSP31/pVAX1重组表达质粒,免疫BALB/c小鼠,观察其免疫应答及免疫保护效果。方法克隆BCSP31基因,构建BCSP31/pVAX1重组表达质粒。转染COS7细胞,免疫组化检测其BCSP31蛋白抗原的表达。BCSP31/pVAX1重组表达质粒肌肉注射免疫BALB/c小鼠,观察特异性体液免疫和细胞免疫效果。进一步用1.25×104布氏杆菌A544强毒株腹腔攻毒,观察BCSP31/pVAX1重组表达质粒的免疫保护效果。结果扩增的BCSP31保护性抗原基因片断在牛、羊和猪布氏杆菌株中具有高度保守性,构建的BCSP31/pVAX1重组表达质粒在COS7细胞有目的蛋白抗原的表达。制备的BCSP31/pVAX1重组表达质粒肌肉免疫BALB/c小鼠4次,ELISA检测BCSP31/pVAX1重组表达质粒产生了较高水平的特异性IgG抗体,并随免疫次数增加抗体的滴度也递增,且抗体亚型以IgG2a为主,表明主要引起Th1型的免疫应答。FCM检测BCSP31/pVAX1重组表达质粒的CD4+/CD8+比值明显下降,说明激发了显著的CTL效应。MTT测定BCSP31/pVAX1重组表达质粒的淋巴细胞增殖能力与对照组比较差异显著。布氏杆菌A544强毒株攻毒后,动物存活及脾组织细菌数与对照组比较有显著性差异,说明BCSP31/pVAX1重组表达质粒能够产生有效的保护效果。结论研制的BCSP31/pVAX1重组表达质粒免疫BALB/c小鼠能产生高水平的特异性体液免疫和细胞免疫,以细胞免疫为主,并产生有效的免疫保护效果,为布氏杆菌病新型疫苗的研究奠定了基础。

Construction of the recombinant expression plasmid BCSP31/pVAX1 of Brucella and evaluation of its immuno-protective effect

To construt the recombinant expression plasmid BCSP_ 31/pVAX1 of Brucella and to evaluate its immuno-protective effect, the Brucella BCSP_ 31 gene was cloned into eukaryotic expression plasmid PVAX1 in order to construct the recombinant plasmid BCSP_ 31/pVAX1, which was then transfected into COS-7 cells.A large amount of plasmid was used to immunize BALB/c mice, and the protective effect was evaluated by comparing the level of infection in the spleens of the vaccinated mice challenged with B.abortus A544 strain.The experimntal results showed that the recombinant plasmid could be expressed in the COS-7 cells with a typical T-helper 1-dominanted immune response as demonstrated by IgG isotype analysis. Meanwhile, high titers of the specific antibodies against BCSP_ 31 protein were detected by ELISA, and the Con-A induced proliferation of the splenic T lymphocytes in the immunized mice was increased as shown by the T lymphocyte assay.Results in flowcytometry analysis showed a lower CD_ 4+/CD_ 8+ ratio in comparison with that of the control group. It was also found that immunization of mice with rcombinant plasmid BCSP_ 31/pVAX1 reduced the bacterial burden relative to that in the control group.Altogether, these data suggest that recombinant plasmid BCSP_ 31/pVAX1is a good immunogen for the production of humoral and cell-mediated immune responses in mice and would be a better candidate for use in the future studies of vaccination against brucellosis