牛新孢子虫NcSAG1基因工程亚单位疫苗的免疫试验

为了解新孢子虫NcSAG1表面蛋白基因工程亚单位疫苗对实验动物的免疫效果,本试验提取延边黄牛新孢子虫野毒株基因组DNA,用PCR技术扩增新孢子虫NcSAG1表面蛋白基因,并在大肠杆菌中进行原核表达,将Western-blot鉴定具有免疫活性的重组蛋白与弗氏佐剂混合制备NcSAG1基因工程亚单位疫苗,免疫BALB/c小鼠,应用间接ELISA方法测定体液免疫水平,应用流式细胞技术测定细胞免疫水平,以此评价疫苗对实验动物的免疫应答反应。结果显示,制备的NcSAG1基因工程亚单位疫苗免疫BALB/c小鼠后,在三免后第3天时,检测抗体的OD450nm值达0.688;CD4+/CD8+值达3.650,均显著高于重组蛋白免疫组和PBS对照组,说明制备的基因工程亚单位疫苗能够提高实验动物的体液免疫和细胞免疫水平。本试验为牛新孢子虫NcSAG1表面蛋白基因工程亚单位疫苗的深入研究奠定了基础。

Immunity assay of the NcSAG1 subunit vaccine against bovine Neospora caninum

To investigate the immunity effect of the surface protein NcSAG1 subunit vaccine in experimental animals, genomic DNA of bovine Neospora caninum wild strain in Yanbian was extracted, and used to amplify NcSAG1 gene by PCR. The PCR product was cloned and expressed in E.coli. NcSAG1 subunit vaccine was prepared by mixing Freund′s adjuvant (FA) with the immunologically competent recombination protein identified by Western-botting. Balb/c mice were immunized with the vaccine. The level of humoral immunity was determined by indirect ELISA. The level of cell mediated immunity was determined by flow cytometer. The immune response of the vaccine in experimental animals was estimated. The result showed that on the third day after Balb/c mice were immunized by NcSAG1 subunit vaccine, OD450nm value of the antisera was up to 0.688 and CD4+/CD8+ was 3.650, markedly higher than those in the immunization group with the single recombination protein and PBS control group. It suggested that the subunit vaccine could improve humoral and cell mediated immunity levels of experimental animals. This study lays a foundation for the clinical application of the NcSAG1 subunit vaccine against bovine Neospora caninum.