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Study on the antibody production and detection to the major outer membrane protein of Vibrio anguillarum in grouper
|Authors: ||Shui-Tsung Chen|
|Contributors: ||NTOU:Department of Environmental Biology and Fisheries Science|
Vibrio anguillarum;major outer membrane protein;antigen;antibody;polyacrylamide;gel electrophoresis;western blot;immunochemistry assay
|Issue Date: ||2011-06-30T08:19:00Z
|Abstract: ||由於台灣養殖漁業密集飼養，造成養殖魚類感染性疾病劇增，使得養殖魚貝類大量死亡，直接影響到國家重大經濟損失。其中魚類感染性疾病的病原體，以弧菌所造成疾病最為普遍，因此有關水產養殖魚類的弧菌症的研究備受重視，於是專家學者針對此弧菌的致病原因、致病機制及病原體診斷、疫苗開發進行研究探討。至今發現鰻弧菌有十種以上的O抗原，其中以O1、O2、O3三種血清型為最常見，在不同血清型鰻弧菌中其菌體的外膜皆具有一種共同主要外膜蛋白，而對主要外膜蛋白在鰻弧菌感染魚體的病原體診斷與疫苗開發上所代表分子角色值得研究開發，於是本論文根據此動機進行研究，首先是將萃取O1、O2、O3血清型鰻弧菌的主要外膜蛋白經蛋白質電泳分析，O1為41 KDa、O2為35 KDa、O3為37 KDa，分別以此當成抗原，進行兔子抗體的製備。我們根據各主要外膜蛋白相互之間，抗原與抗體之間交叉反應，經西方氏轉漬法與免疫化學染色法來檢測，得知O2與O3之間有明顯交叉反應，而 O1與O2、O3之間無交叉反應，由此推知O2、O3血清型鰻弧菌變異性相似，O1血清型鰻弧菌與O2、O3血清型相異。接著我們直接使用，經福馬林去活化的死鰻弧菌或活鰻弧菌注射於石斑魚進行抗體試驗，感染後魚體所產生的抗體可與O1、O2、O3血清型主要外膜蛋白的抗原產生反應。由此結果得知，主要外膜蛋白分子未來也許可作為一種正確、精準的早期診斷鰻弧菌的病原體診斷試劑開發的依據，甚至可作為鰻弧菌次單元疫苗的抗原，進而達到鰻弧菌在石斑魚的預防，以減少石斑魚養殖的損失。|
In Taiwan, the disease rate of farming fish has increased tremendously due to the way of high-density farming fishery, which resulted in high death rate of fishes and shells. The economic consequence has been devastating. Since vibrosis is the most commonly-encountered infectious diseases in fish, research related to vibrosis of farming fish has been intensively carried out worldwide. Researches include diseases caused by vibrio bacteria, pathogen recognition and detection, and vaccine development. Vibrio anguillarum is a Gram-negative bacterium which is classified into 10 serotypes based on its liposaccharides O antigens. In addition to the O-antigen, the major outer membrane protein (MOMP) of V. anguillarum has also been identified as a major antigenic determinant. Among these serotypes, O1, O2, and O3 serotypes are the most commonly found. We believed that the MOMPs of V. anguillarum may act as the targets for diagnosis and vaccine development. Their molecular masses are 41, 35, and 37 kDa for O1, O2 and O3 MOMPs, respectively. In this work the MOMPs of O1, O2 and O3 serotypes of V. anguillarum were individually purified by polyacrylamide gel electrophoresis (PAGE). Each has been used as antigen to elicit antibody from rabbit. The cross reaction of produced antibodies with those three antigens were checked by Western blot and immunochemistry assays. There is cross reaction between O2 and O3, but O1 has no cross reaction with O2 and O3. These results are consistent with the previous known serotypical properties among the three serotypes. Antibodies were found in grouper injected with either formalin-treated (inactivated) or live V. anguillarum and they reacted with the corresponding MOMPs of O1, O2 and O3 serotypes. Our results suggest that the MOMPs might be used as the diagnostic reagent for early detection of V. anguillarum infection in fish. They may be further tested for vaccine development in the future.
|Appears in Collections:||[環境生物與漁業科學學系] 博碩士論文|
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