猪丹毒丝菌SpaA基因原核表达及表达蛋白质的免疫原性分析

发布时间：2018-12-29 13:54

【摘要】：旨在克隆表达猪丹毒丝菌SpaA蛋白,分析其免疫原性,为猪丹毒新型疫苗的研发奠定基础。运用PCR扩增SpaA基因,连接至pGEX-6P-1载体,将阳性重组质粒转化大肠杆菌Rosetta(DE3),IPTG诱导表达,SDSPAGE分析表达产物,GST亲和层析纯化融合蛋白质,Western blot鉴定;纯化SpaA蛋白免疫小鼠,间接ELISA检测血清中IgG和Th1、Th2相关细胞因子;不同致病力猪丹毒丝菌攻击免疫小鼠,观察病理组织变化及其免疫保护率。结果显示,猪丹毒丝菌SpaA基因在大肠杆菌中实现表达,获得大小为75ku的纯化目的蛋白质。该蛋白质能特异性识别猪丹毒丝菌抗血清,可诱导小鼠产生较高水平的特异性抗体IgG,并使TNF-β、IFN-γ、IL-5、IL-10含量显著升高,对攻毒菌的免疫保护率均为100%,病理组织变化与攻毒对照组之间差异明显。可见成功获得的重组SpaA蛋白具有良好的免疫原性,能激发机体的体液免疫和细胞免疫,产生较强的免疫保护力,可以作为研发猪丹毒亚单位疫苗的候选抗原。
[Abstract]:The aim of this study was to clone and express the SpaA protein of porcine erysipelas and analyze its immunogenicity so as to lay a foundation for the research and development of a new vaccine of porcine erysipelas. The SpaA gene was amplified by PCR and ligated to the pGEX-6P-1 vector. The positive recombinant plasmid was transformed into E. coli Rosetta (DE3), IPTG induced expression, SDSPAGE analysis, GST affinity chromatography). The fusion protein, Western blot was purified by GST affinity chromatography. The mice were immunized with purified SpaA protein, the serum IgG and Th1,Th2 related cytokines were detected by indirect ELISA, the mice were immunized with different pathogenicity swine erysipelas, and the pathological changes and the immune protective rate were observed. The results showed that the SpaA gene of erysipelas porcine was expressed in Escherichia coli and purified protein with the size of 75ku was obtained. The protein can specifically recognize the antiserum of Rhizoma erysipelas, induce the production of high level of specific antibody IgG, and increase the contents of TNF- 尾, IFN- 纬 and IL-5,IL-10 in mice. There was a significant difference between the pathological changes and the control group. It can be seen that the recombinant SpaA protein obtained successfully has good immunogenicity, can stimulate humoral and cellular immunity and produce strong immune protection, and can be used as a candidate antigen for the development of porcine erysipelas subunit vaccine.
【作者单位】：安徽农业大学动物科技学院;安徽省畜禽产业共性技术研究院;
【基金】：国家星火计划重点项目(2014GA710002) 安徽省质量工程项目(2013sxzx008) 安徽省生猪产业体系基金
【分类号】：S852.6

【参考文献】