以减毒沙门菌为载体布鲁氏菌DNA疫苗的构建及免疫原性分析

Construction of a recombinant attenuated Salmonella typhimurium DNA vaccine carrying brucella BLS-L7/L12 fusion gene

目的构建携带布鲁氏菌BLS-L7/L12融合基因的重组减毒沙门菌并进行免疫原性分析,为口服布鲁氏菌DNA疫苗研究奠定基础。方法将BLS-7/L12融合基因克隆到真核表达载体asd+-pVAX1,依次将重组质粒转化减毒沙门菌X3730、X4550得到重组沙门菌X4550(asd+-pVAX1-BLS-L7/L12)。以1×109CFU/只的剂量口服免疫Balb/C小鼠,3次免疫后进行免疫效果的评价。结果构建的重组减毒沙门菌质粒转染COS-7细胞经免疫组化和Western-blot试验证明BLS-L7/L12融合蛋白在细胞中得到了瞬时表达,ELISA检测到免疫小鼠血清和肠黏液中有特异性抗体IgG和sIgA产生。通过淋巴细胞增殖实验、细胞因子和CD分子测定表明DNA疫苗以诱发Th1型免疫为主。结论所构建的以重组沙门菌为载体口服布鲁氏菌DNA疫苗具有诱导特异性细胞免疫和体液免疫应答的能力,且以细胞免疫应答为主。可作为潜在的布鲁氏菌新型疫苗。

Objective To construct a recombinant attenuated Salmonella typhimurium DNA vaccine carrying bruceUa BLS-L7/L12 gene and to detect its immunogenicity. Melhods Brucella BLS-L7/L12 gene was cloned into the eukaryotic expression vector asd^＋ -pVAX1, and then the recombinant plasmid was used to transform Escherichia coli X6212, attenuated S. typhimurium X3730, and X4550. After that, the recombinant strain was inoculated into Balb/c mice by oral gavage with 109 CFU. Results Recombinant expressing plasmid asd^＋ -pVAX1- BLS-L7/L12 （pVa-BL） was constructed successfully. The BLS-L7/L12 protein was expressed in attenuated S. typhimurium highly and stably. Animals immunized with S. typhimurium （pVa-BL） both induced antigen specific antibody and typical Thl-dominated immune response, as determined by lymphocyte proliferation assay, cytokine and immunoglobulin Gisotype analysis. Conclusion S. typhimurium （pVa-BL） can induce both humoral and cellular immune response. This vaccine could be used as a potential candidate vaccine for the control of brucellosis.