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Immunological and Biochemical Studies on a Hemolysin Produced by Aeromonas Salmonicida

杀鲑气单胞菌产生的溶血素的免疫学和生化研究

基本信息

批准号：
63560196
负责人：
NOMURA Setsuzo
金额：
$ 1.6万
依托单位：
Kitasato University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1988
资助国家：
日本
起止时间：
1988 至 1990
项目状态：
已结题

项目摘要

The 50 % lethal doses (LD_<50>) of purified salmolysin for white-spotted char on intramuscular and intraperitoneal injection were 196 and 556 mu/kg. The LD_<50> for rainbow trout on intramuscular injection was 58 mu/kg. Salmolysin was detoxificated by heat-treatment at 60 ^0C for 30 min, or by formalin-treatment for 1 week. Salmolysin toxoid was injected to white-spotted char intramuscularly. As the result, agglutinating antibody titer was reached to maximum after 3 weeks. White-spotted char were immunized with salmolysin toxoid by intramuscular injection or immersion method, after, that, the fish were cahllenged with Aeromonas salmonicida. AS the result, survival ratio of immunized fish was higher than that of non-immunized fish. These results indicate that salmolysin toxoid is an effective immunogen against furunculosis. Salmolysin antitoxin was purified from the serum of white-spotted char by ammonium sulfate precipitation, anion-exchange chromatography on DEAE-cellulose, and gel filtration on Sephatose 6B. Furthermore, salmolysin produced in the culture supernatant of A. salmonicida and in the focus tissue of infected fish was detected by enzyme linked immuno-sorbent assay. On the other hand, the red blood cells of salmonids were peculiarly lysed by salmolysin, however, the red blood cells of fowls and mammalia were not lysed by the toxin. Since the sensitivity of salmonid red blood cells to the toxin was different, it was suggested that the surface structure of salmonid erythrocytes varies with the genus level. Salmolysin did not show phospholipase C activity and was not reactivated by thiolcompound. The hemolytic catibity of salmoysin was not affected by phospholipids, however, the activity was stimulated by asialoglycophorin.

纯化<50>的沙门氏菌蛋白酶对白斑红点鲑肌肉注射和腹腔注射的半数致死量分别为196和556 μ/kg。肌肉<50>注射对虹鳟鱼的LD_（58 μ g/kg）。沙门氏菌蛋白酶经60 ℃热处理30分钟或福尔马林处理1周后可脱毒。将沙门氏菌蛋白酶类毒素肌肉注射到白斑红点鲑。结果，凝集抗体滴度在3周后达到最大值。用沙门氏菌蛋白酶类毒素肌肉注射或浸泡法免疫白斑红点鲑，然后用杀鲑气单胞菌进行人工免疫。结果表明，免疫鱼的存活率高于未免疫鱼。这些结果表明，沙门氏菌蛋白酶类毒素是一种有效的抗呋喃丹的免疫原。通过硫酸铵沉淀、DEAE-纤维素阴离子交换层析和Sephatose 6 B凝胶过滤，从白点炭血清中纯化了鲑鱼蛋白酶抗毒素。此外，A.用酶联免疫吸附试验检测病鱼病灶组织中的杀鲑素和杀鲑素。沙门氏菌蛋白酶对鲑科动物的红细胞有特异性的溶解作用，而对禽类和哺乳动物的红细胞无溶解作用。由于鲑鱼红细胞对毒素的敏感性不同，因此推测鲑鱼红细胞的表面结构因属而异。Salmolysin没有表现出磷脂酶C活性，也没有被巯基化合物重新激活。溶血素的溶血活性不受磷脂的影响，但能被脱唾液酸血型糖蛋白激活。