Comparative Study On The Immunological Characteristics Of Primary Pathogenic Bacteria On Aquatic Animals

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.