抗伤寒沙门菌SOD血清的制备及其免疫保护作用的研究

Preparation of anti-Salmonella typhi Fe-SOD and its immuno protection

目的 制备抗伤寒沙门菌Fe SOD血清 ,研究其免疫保护作用 ,探讨SOD与沙门菌毒力的关系。方法 用纯化的伤寒沙门菌 (Stw1)Fe SOD免疫家兔制备兔抗Fe SOD血清。利用ELISA法检测抗血清的效价。免疫电泳、双向琼脂扩散试验和抗体对酶活性抑制试验分析抗血清的特性。用不同浓度的抗血清预先处理Stw1,然后进行小鼠腹腔吞噬细胞的吞噬杀菌试验 ,并利用鼠伤寒沙门菌感染小鼠模型证明抗SOD血清的被动免疫保护作用。结果 抗血清的ELISA效价为 1∶10 2 40 ,免疫电泳结果显示纯化的Fe SOD及伤寒沙门菌无细胞溶解物与抗Fe SOD血清在相同位置各形成一条沉淀线。双向琼脂扩散试验结果显示该抗血清与牛红细胞SOD不形成沉淀线 ,抗血清对酶活性抑制试验结果显示抗血清可抑制伤寒沙门菌及鼠伤寒沙门菌无细胞溶解物中部分SOD活性 ,对牛红细胞SOD的抑制率较低。小鼠腹腔吞噬细胞对伤寒沙门菌的吞噬杀菌试验结果显示 ,经中浓度的抗血清处理的细菌在 6 0min、12 0min时和经高浓度抗血清处理的细菌在 30min、6 0min及 12 0min时CFU值明显低于对照组 (P <0 .0 1)。经抗血清处理过的 5LD5 0 鼠伤寒沙门菌攻击的小鼠 3d和 7d的存活率分别为90 %和 6 0 % ,均明显高于对照组 (P <0 .0 5或P <0 .0 1)。

Objective To prepare the anti Salmonella typhi Fe SOD and to study the immuno protection of this antiserum. Methods The anti Fe SOD was raised in the rabbit immunized and then titrated by ELISA. The antiserum were further characterized by immuno eletrophorisis, double agar difusion, enzym activity inhibition and opsonization test with murine peritoneal macrophages. Results The titer of the antiserum was 1∶10240. The cell free extracts and the purified Fe SOD from S.typhi but not the SOD from bovine RBC formed identical precipitation line with the antiserum. The 63.39% 73.38% of SOD activities in cell free extracts from different S.typhi were inhibited by the antiserum as shown in NBT reduction inhibition test, but only 42.59% and 24.92% SOD activity from S.typhimurium and bovine erythrocyte were inhibited, respectively by the antiserum(P<0.01 or P<0.05). The bacteria number of test groups where 1ml or 2ml of the anti SOD were added were both much less than that in the control groups at 30?min, 60?min and 120?min, respectively in a phagocytic killing test by murine peritoneal phagocytes (P<0.01). The survival rates of mice in the group challenged with 5LD 50 S.typhimurium incubated with anti Fe SOD serum were 90% and 60% during a period of 3 days and 7 days, respectively. Conclusion The results showed that the anti Fe SOD could not only strengthen the phagocytic killing of the bacteria by murine peritoneal phagocytes in vitro but also offers cross immuno protection to the mice challenged with S.typhimurium. The SOD seems to function as common protective antigen of Salmonella.