作者：徐军发,唐湘涓        单位：广东医学院检验科

关键词：伤寒沙门菌；鼠伤寒沙门菌；Fe-SOD；抗血清；免疫保护作用

【 摘要 】　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∶10240，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60min、120min时和经高浓度抗血清处理的细菌在30min、60min及120min时CFU值明显低于对照组（P＜0.01）。经抗血清处理过的5LD50鼠伤寒沙门菌攻击的小鼠3d和7d的存活率分别为90%和60%，均明显高于对照组（P＜0.05或P＜0.01）。 结论　抗伤寒沙门菌SOD血清可促进小鼠腹腔吞噬细胞对伤寒沙门菌的杀灭，也能提高鼠伤寒沙门菌攻击小鼠的存活率，说明此抗血清具有免疫保护作用，提示SOD可能是沙门菌共同的保护性抗原，间接说明了伤寒沙门菌的SOD与其毒力有关。

Preparation of anti-Salmonella typhi Fe-SOD

and its immuno-protection

XU Junfa, TANG Xiangjuan. Department of Laboratory Medicine, Guangdong Medical College, Zhanjiang, P.R.China

【 Abstract 】　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 30min, 60min and 120min, respectively in a phagocytic killing test by murine peritoneal phagocytes (P＜0.01). The survival rates of mice in the group challenged with 5LD50 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.

【 Subject words 】　S.typhi; S.typhimurium; SOD; Antiserum; Immuno-protection

吞噬细胞呼吸爆发过程中产生的超氧阴离子（O2）、过氧化氢（H2O2）、羟自由基（OH*）和单线态氧（1O2）是重要的杀微生物物质〔1〕。病原微生物，尤其是细胞内寄生的细菌具有多种抗吞噬细胞杀伤的机制，其中超氧化物歧化酶（superoxide dismutase，SOD）、过氧化氢酶（catalases，CAT）、过氧化物酶（peroxidase，POD）等可分解吞噬细胞产生的活性氧，从而保护细菌逃避吞噬细胞的杀伤。因此，SOD、CAT、POD被认为是某些细菌重要的毒力因子〔1〕。已有报道，星形诺卡菌〔2，3〕 、结核分枝杆菌〔4〕、福氏志贺菌〔5〕、大肠杆菌〔6〕等的SOD与其毒力有关。星形诺卡菌SOD抗体能增强吞噬细胞对该菌的吞噬杀菌作用〔2,3〕。关于沙门菌SOD与其毒力的关系目前未见报道。本研究制备了兔抗伤寒沙门菌(Stw1) Fe-SOD血清，并对其免疫保护作用进行了研究。