布氏杆菌PBP_(39)/pCDNATE重组表达质粒的构建及免疫效果的研究

Construction of recombinant expressing plasmid of PBP_(39)/pCDNATE and evaluation of its immune protective effection

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。

Objective To construct recombinant expressing plasmid for evaluating the humoral immune response, cellular immune response and immune protection. Methods Brucella PBP39 gene was cloned into the eukaryotic expressing plasmid pCDNATE to get a recombinant plasmid pCDNATE-PBP39, which was then transfected into Cos-7 cells. A large amount of the plasmid were used to immunize BALIVc mice. Protection was evaluated by comparing the levels of infection in the spleens of vaccinated mice challenged with B . abortus 5 4 4 . Results 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 fiter specific antibody against PBP39 protein was detected by ELISA. T lymphocyte assay showed that the Con A-induced proliferation of spleenic T lymphocytea in the immunized mice was increased. Immunization with pCDNATE-PBP39 reduced the bacterial burden relative to those in the control groups. Conclusion pCDNATE-PBP39 is a good immunogen for the production of cell-mediated responses in mice and a candidate for vaccine against brucellosis.