Trap抗原诱导细胞免疫对S.aureus免疫保护作用的研究

Study on the Protective Effect of Trap Antigen-induced Cellular Immunity on S.aureus

金黄色葡萄球菌(S.aureus)是奶牛乳房炎的主要致病菌。实验利用S.aureus的Trap基因构建Trap/PET-32a表达质粒,IPTG诱导表达后柱层析纯化Trap蛋白,与弗氏佐剂混合制备疫苗,小鼠肌肉免疫,二免后7 d,分离小鼠脾细胞,经Trap抗原刺激后检测细胞因子,二免后14 d,小鼠眼球采血制备血清检测抗体,小鼠腹腔S.aureus Newman株攻毒2 d后,取小鼠的肝、脾、肺、肾,涂板检测细菌荷载量,攻毒7 d后制作病理组织切片。结果显示Trap蛋白免疫组比标签蛋白PET-32a对照组极显著增强了细胞因子IFN-γ,IL-4和IL-17A的表达水平与特异性IgG的表达水平;显著降低了细菌对小鼠各个器官的损伤与定植,攻毒保护保护率为56%。实验验证了S.aureus的Trap抗原小鼠免疫既增强了体液免疫又提高了细胞免疫水平,对小鼠有理想的免疫保护作用。

Staphylococcus aureus(S.aureus)is the main pathogen of dairy cow mastitis.In the experiment,the Trap gene of S.aureus was used to construct the Trap/pET-32a expression plasmid,the Trap protein was purified by column chromatography after IPTG induced expression,and the vaccine was prepared by mixing with Freund’s adjuvant.The mice were immunized with muscles.7 days after the second immunization,the mice were isolated.Mouse spleen cells were stimulated by Trap antigen to detect cytokines.14 days after the second immunization,blood was collected from the eyeballs of mice to prepare serum detection antibodies.After 2 days of challenge with S.aureus Newman strain in the abdominal cavity of mice,the liver,spleen,kidney and lungs of the mice were taken.smear plate to detect bacterial load,and make pathological tissue slices 7 days after challenge.The results showed that the Trap protein immunization group significantly enhanced the expression level of cytokines IFN-γ,IL-4 and IL-17A and the specific IgG expression level compared with the label protein pET-32a control group;significantly reduced the bacterial impact on mice.Injury and colonization of organs,the protection rate against poison is 56%.The experiment verified that S.aureus’s Trap antigen mouse immunization not only enhanced humoral immunity but also increased the level of cellular immunity,and had an ideal immune protection effect on mice.