【作者】 齐继光；

【导师】 战文斌；

【作者基本信息】 中国海洋大学 ， 水产养殖， 2002， 硕士

【摘要】 本文主要通过不同的生物及免疫学技术和方法对细菌与不同抗体间的交叉反应进行了实验，并对结果进行分析，探讨一种新的对于细菌性疾病的诊断方法，并且利用这种方法对生产中的多个病例进行了诊断验证，均取得了较好的结果。 将副溶血弧菌，哈维氏弧菌，溶藻胶弧菌，鳗弧菌，爱德华氏菌，荧光假单胞菌进行富集培养，并用其免疫大鼠，得到了六种细菌的多克隆抗血清。以所得抗血清和六种细菌作材料，利用免疫试管凝集法，丙烯酰胺凝胶电泳法，免疫印迹法，荧光抗体法，酶联免疫吸附法等生物学方法和技术对于这些抗体和抗原之间的交叉反应作了详尽的研究和分析，发现了在不同细菌与不同抗体之间交叉反应的特异性是不相同的，这就为我们区分和鉴定细菌提供了依据。 免疫沉淀的实验结果显示，各抗体与各细菌之间存在着较大的交叉反应，其规律是同属之间抗体与细菌的交叉反应较大。四种弧菌之间均存有不同程度的交叉。而荧光假单胞菌几乎不与任何一种弧菌发生反应，与迟钝爱德华氏菌也仅存在着较低程度的交叉。 通过免疫印迹法计算了各细菌对于不同抗体具有抗原性的特异性蛋白条带的分子量。结果显示，不同的菌种其抗原决定簇是不同的，虽在同属间有的菌种交叉的比较大，但是可以直观地看出各细菌抗原决定簇的多少和特异性蛋白带的位置，用Western-blot法鉴定细菌的种类由于可以量化，所以是一种值得深入研究的方法。细菌具有的特异性蛋白可以作为诊断依据之一。 利用免疫荧光法对六种细菌与六种已知抗体的交叉反应进行了检测，反应结果与免疫印迹法和免疫沉淀法的结果基本一致，依然是同属之间的交叉反应程度比较大，爱德华氏菌与荧光假单胞菌之间的交叉反应比较大。 ELIsA是一种灵敏度较高的免疫测定技术，它是以样品的吸光值作为判断阴阳性的标准，客观性较好，能够较准确的说明交叉反应的程度，从El创工SA结果可以看出，弧菌之间交叉反应程度依然是最大的，这与前文的结果基本一致，因此更加说明了，同属之间的抗体与抗原之间的交叉反应较大，并存在具有相同分子量具抗原性的特异蛋白带，这些蛋白带可能是我们诊断鉴定细菌的重要依据。 运用上述所研究的方法对于生产中遇到分离的四株病原菌进行了分析和诊断。通过交叉反应的结果分析，细菌二、三、四为弧菌，细菌一为爱德华氏菌。并利用试验结果指导了用药，所得的效果非常明显。且此诊断方法，操作简单，用时少，花费低，所以在实际生产中有一定的可行性。更多还原

【Abstract】 In this paper, different biologic and immunologic technologies were used to study cross reactions between bacteria and different antisera and discover a new kind of method to differentiate the pathogenesis of bacteria diseases. Then the new method was put into practice to diagnose several cases and obtain good results.Vibrio harveyi, Vibrio alginolyticus, Vibrio anguillarum, Vibrio parahaemolyticus, Edwardsiella tarda and Pseudomonas fluorescens were cultured on medium corresponding to the bacteria respectively, then were used to immunize mouse to obtain polyclonal antisera. By use of the technologies of Immediate Agglutination, SDS-PAGE, Western-blot, IFAT and ELISA, the author did detailed research to analyse the cross reactions between pathogenesis and antisera above-mentioned, finding that the level of cross reactions was not the same between different bacteria and different antisera. This supplies a basis for us to differentiate bacteria.The results of agglutination showed that cross reactions existed generally. The level of cross reactions was high when a bacterium react with a antiserum obtained from a bacterium of the same genus as it, or the level was low. There was a high level of cross reactions among the Vibrio, meanwhile Pseudomonas fluorescens could hardly react with any Vibrio, and the cross reaction between Pseudomonas fluorescens and Edwardsiella tarda was in a low level.Through Western-blot the author calculated the molecular weight of specific protein belt with antigenicity when a bacterium had cross reaction with a antiserum. The results showed that different bacteria had different antigenic determinant,although sometimes the bacteria of the same genus had a high level of cross reactions,we still could tell the numbers and sites of the special protein belts. These protein belts can be used as important basis to determine bacterial pathogen. Because it could be quantified using the method of Western-blot to determine the species of bacteria, Western-blot is worthy of being researched deeply.The fluorescence intensity varies with the level of cross reaction, the level of cross reaction between six strains and six antisera was determined, showing no difference with Western-blot and agglutination.ELISA is a high-sensitivity immunoassay technology, it determines the positive or negative of a reaction by reading the OD(optical density) value. It’s an objectivity technology which can tell the level of cross reaction accurately. From the result it could be found that the level of cross reaction between Vibrios was high, it was the same as before. So it could be said that the level of cross reaction among the bacteria of the same genus is high. And the bacteria of the same genus have the specificprotein belts with the same molecular weight. These protein belts can be used as important basis to determine bacterial pathogen.Four strains of bacteria were separated from turbots. These four strains were determined by using the methods above, showing that strain 1 was Edwardsiella, the other three strains were Vibrio. Then the conventinonal method such as drug-fast test was used to examine the strain 1 and obtained the same result.Immunoassay methods can operate easily and cost little time and money, it’s feasible in practice.更多还原