布氏杆菌PBP39/pCDNATE重组表达质粒的构建及免疫效果的研究

目的构建pBp39/pCDNATE重组表达质粒,免疫BALB/c小鼠观察诱导的特异性体液免疫、细胞免疫及免疫保护效果.方法克隆PBP39基因,构建PBP39/pCDNATE重组表达质粒.首先转染Cos-7细胞,免疫组化检测其PBP39蛋白抗原的表达.然后用PBP39基因疫苗肌肉注射免疫BALB/c小鼠4次,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544A强毒株腹腔攻毒,观察PBP39重组表达质粒的免疫保护效果.结果扩增的PBP39保护性抗原基因片段在牛、羊和猪布氏杆菌株中具有高度保守性,构建的PBP39/pCDNATE重组表达质粒在Cos-7细胞中有目的蛋白的表达.制备的PBP39重组表达质粒肌肉免疫BALB/c小鼠4次,ELISA检测PBP39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,抗体亚型以IgG2a为主,表明诱导了TH1型的免疫应答.MTT测定PBP39重组表达质粒的淋巴细胞增殖能力与对照组差异有统计学意义.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差异有统计学意义,说明PBP39重组表达质粒能够产生有效的保护效果.结论研制的PBP39重组表达质粒免疫BALB/c小鼠能产生高水平的特异性抗体和细胞免疫,且以细胞免疫为主,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 recombinant expressing plasmid of PBP39/pCDNATE and evaluation of its immune protective effection

Objective To construct recombinant expressing plasmid for evaluating the humoral immune response, cellular immune response and immune protection. Methods Brucella PBP_ 39 gene was cloned into the eukaryotic expressing plasmid pCDNATE to get a recombinant plasmid pCDNATE-PBP_ 39 , which was then transfected into Cos-7 cells. A large amount of the plasmid were used to immunize BALB/c mice. Protection was evaluated CM(43]by comparing the levels of infection in the spleens of vaccinated mice challenged with B.abortus 544. ResultsCM)] The recombinant plasmid can be expressed in Cos-7 cells. The typical T helper 1 dominated immune response, as determined by immunoglobulin G isotype analysis. The high titer specific antibody against PBP_ 39 protein was detected by ELISA. T lymphocyte assay showed that the Con A-induced proliferation of spleenic T lymphocytes in the immunized mice was increased. Immunization with pCDNATE-PBP_ 39 reduced the bacterial burden relative to those in the control groups. Conclusion pCDNATE-PBP_ 39 is a good immunogen for the production of cell-mediated responses in mice and a candidate for vaccine against brucellosis.