【作者】 刘洪明；

【导师】 周丽； 战文斌；

【作者基本信息】 中国海洋大学 ， 水产养殖， 2003， 硕士

【摘要】 本文对水产养殖动物6种主要病原菌，哈维氏弧菌(Vibrio harveyi)、溶藻胶弧菌(V. alginolyticus)、鳗弧菌(V. anguillarum)、副溶血弧菌(V. parahaemolyticus)、迟钝爱德华氏菌(Edwardsiella tarda)和荧光假单胞菌(Pseudomonas fluorescens)的全菌蛋白及外膜蛋白的结构和免疫特性作了比较分析。发现同属与非同属细菌之间，这些蛋白的结构差异有一定的规律可循，而这些规律将有助于我们对细菌鉴定的研究和对高效疫苗的开发。 本研究利用免疫试管凝集法，聚丙烯酰胺凝胶电泳法，免疫印迹法，荧光抗体法，酶联免疫吸附法等对6种病原性细菌的全菌蛋白进行了结构和免疫特性的分析。其结构主要通过聚丙烯酰胺凝胶电泳法电泳图谱形式表现；而各个细菌的免疫特性则由细菌与其抗体之间的交叉反应来实现。通过上述5种方法对其交叉性的测定发现，同属之间的抗原抗体的交叉反应较大。并且通过Western-blot还可以看出同属细菌之间交叉反应的蛋白带，多数具有相同的分子量。实验证明：抗哈维氏弧菌的抗体，与哈维氏弧菌，溶藻胶弧菌和副溶血弧菌有明显的交叉反应，分子量相同的交叉反应蛋白带为：135.6kDa，121.5kDa，而此抗体与迟钝爱德华氏菌和荧光假单胞菌在此蛋白处没有交叉反应。抗溶藻胶弧菌的抗体，与哈维氏弧菌，溶藻胶弧菌和副溶血弧菌有明显的交叉反应，分子量相同的交叉反应蛋白带为：96.5kDa，48.4kDa，39.2kDa，34.9kDa，而此抗体与迟钝爱德华氏菌和荧光假单胞菌在此蛋白处没有交叉反应。抗副溶血弧菌，与哈维氏弧菌，鳗弧菌和副溶血弧菌，分子量交叉反应的蛋白带为：55.1kDa，而此抗体与迟钝爱德华氏菌和荧光假单胞菌在此蛋白处没有交叉反应。弧菌间出现的免疫交叉反应的蛋白带可能是弧菌对于抗弧菌抗体所具有的特异性的蛋白。可以作为诊断弧菌病的依据之一。 本研究还以迟钝爱德华菌为材料分析比较了PMSF、Triton X-100、SDS及Sarkosyl四种细菌外膜蛋白分离制备方法对水产病原细菌的主要外膜蛋白(MOMP)组成结构的影响。结果表明PMSF与Triton X-100方法制备的迟钝爱德华氏外膜蛋白，SDS-PAGE电泳显示多条蛋白带，其主要外膜蛋白分子量为114kDa。和50kDa；SDS和Sarkosyl方法，主要外膜蛋白带有4条，大致分子量为67kDa、水产动物主要病原细菌免疫学特性的比较研究64 kDa、50 kDa和37 kDa。与兔抗迟钝爱德华氏菌血清的Western一blotting印迹显示抗血清与迟钝爱德华氏菌菌株的外膜蛋白有2条免疫反应带，其大致分子量为114kDa和37kDa。通过比较分析认为，Sarkosyl法对提取水产病原性细菌外膜蛋白是较为有效的。 为探寻水产病原弧菌间的共同抗原决定簇以及弧菌本身的特异性抗原决定簇，为水产动物细菌性病原的早期快速诊断以及弧菌亚单位疫苗和DNA疫苗提供基础材料。本研究对溶藻胶弧菌和鳗弧菌全菌、破碎菌及外膜蛋白的免疫特性作了初步的分析。结果证明了它们结构之间存在着差异:溶藻胶弧菌的外膜蛋白主要有五种:26kDa，37kDa，40kDa，43kDa，47kDa;鳗弧菌的外膜蛋白主要有六种:20kDa，37kDa，45kDa，49kDa，58kDa，64kDa。免疫印迹显示鳗弧菌的发色带主要有一条，分子量为:49kDa。溶藻胶弧菌的发色带主要有两条，分子量分别为:26kDa，43kDa。在外膜上，通过这种方法没有找到这两种弧菌之间没有共的抗原决定簇。可能这两种细菌之间在外膜蛋白上没有相同的抗原决定簇，本研究将会对更多种弧菌的外膜蛋白进行比较，以期找出几种弧菌所共同具有的抗原决定簇。更多还原

【Abstract】 Analyses and comparative studies on the structures and immunological characteristics of out membrane proteins (OMPS) extracted from Vibrio harveyi, V. alginolyticus, V. anguillarum, V. parahaemolyticus ,Edwardsiella tarda and Pseudomonas fluorescens were carried out. The results show that the regulations between the structures of OMPS in the same category and in different ones. These regulations will help us on the study of bacterial identifying and vaccine developing.With Immediate Agglutination, SDS-PAGE, Western-blot, IFAT and ELISA methods, the analyses were carried out on structures and immunological characteristics of the whole proteins possessed by the 6 bacteria mentioned above. The structures they have can be showed through the SDS-page atlas. And the immunological characteristics can be showed through the cross-reactions between pathogenesis and antiserums. From the results of western-blot, we can conclude that many protein belts of the bacteria in the same category always have the same molecular weight. To anti- Vibrio harveyi serum, Vibrio harveyi, Vibrio alginolyticus, Vibrio parahaemolyticus all have two protein belts with the same molecular weight, 135.6kDa, 121.5kDa, and we can’t find the two protein belts in Edwardsiella tarda and Pseudomonas fluorescens. To anti- Vibrio alginolyticus serum, Vibrio harveyi, Vibrio alginolyticus, Vibrio parahaemolyticus all have four protein belts with the same molecular weight, 96.5kDa, 48.4kDa, 39.2kDa and 34.9kDa. The level of cross-reaction between anti- Vibrio anguillarum serum and Vibrios is too high to differentiate the cross-recreation-protein belt. To Vibrio parahaemolyticus, except Vibrio alginolyticus, the other three Vibrios all have the protein belt 55.1kDa that bacteria of other category don’t have. These maybe the characteristic protein of Vibrio reacting with anti-Vibrio serum, and can be used as one of the basis to diagnose the disease caused by vibrios.To analyze the extraction influences on the structures of bacterial major outer membrane protein (MOMP), four different extraction methods were used for preparing MOMP from the cell membrane fraction of Edwardsiella tarda. PMSF and Triton X-100 extracted many proteins, including protein bands at 114 kDa and 50 kDa in SDS-PAGE. Sodium dodecyl sulfate (SDS) and sodium lauryl sarcosinate (Sarkosyl), however, extracted four MOMPs at 67kDa, 64kDa, 50kDa and 37kDa. These MOMPs were distinctly detected in the Western blotting with rabbit antiserum against Edwardsiella tarda whole cell, indicating the 114kDa and the 37kDa protein are involved in the determination of the cell antigenicity. All the results show that theSarkosyl method is the better one to extract the OMPs from the aquatic bacteria.To find out the uniform and specific epitopes possessed by vibrios and prepared for fast diagnose and vaccine development, SDS-PAGE and WESTERN-BLOT were used in this paper to compare the differences of outer membranes of Vibrio anguillarum and Vibrio alginolyticus on the immunological characteristics. The former one reveals 6 bands: 20kDa, 37kDa, 45kDa, 49kDa, 58kDa, 64kDa, and the latter 5 binds: 26kDa, 37kDa, 40kDa, 43kDa, 47kDa. The result of Western-blot shows that protein 49kDa of Vibrio anguillarum and protein 26kDa, 43kDa of Vibrio alginolyticus can response to the corresponding antibody serum. No uniform epitopes were found out in this study, and we hope that we can find them through more studies on vibrio OMPs.更多还原