本文選題：副溶血弧菌 + 耐熱溶血毒素��； 參考：《上海海洋大學》2011年碩士論文

【摘要】：本文提取與純化副溶血弧菌(Vibrio parahaemolyticus,Vp)耐熱溶血毒素(thermostable direct hemolysin,TDH),對其相關性質進行了分析,通過雜交瘤技術成功制備出TDH的鼠性單克隆抗體,并對其性質進行了鑒定。本文利用副溶血弧菌胞外分泌耐熱溶血毒素這一特性,在含有一定營養(yǎng)成分的細菌培養(yǎng)液增殖培養(yǎng)致病性副溶血弧菌,鹽析提取粗蛋白,之后經過離子交換層析及凝膠多次層析方法對副溶血弧菌耐熱溶血毒素進行了純化,最終純化得到了副溶血弧菌主要致病因子TDH。 由于下面要利用TDH免疫動物,研究探索免疫學方法檢測副溶血弧菌方法,接下來從變性溫度、毒性、耐熱性等幾個理化相關性質進了分析和鑒定。結果表明,副溶血弧菌直接耐熱溶血毒素是典型的蛋白質;此毒素具有較強的毒性效應,少量TDH靜脈注射小鼠就能是小鼠在短時間內死亡;同時,本研究還利用了差示熱量掃描法和加熱處理方法對TDH的耐熱性進行了分析,證明了此毒素具有較強的耐熱性。 實驗中,我們將TDH作為免疫原免疫小鼠成功得到多克隆免疫血清,根據Western斑點印跡原理檢測致病性副溶血弧菌,取得了良好的效果。探討了特異性免疫血清的最佳工作濃度以及分別對致病性副溶血弧菌與非致病性副溶血弧菌的敏感性,結果表明此方法可針對性的檢測分泌TDH的致病性副溶血弧菌,對非致病性副溶血弧菌不敏感,操作簡單、靈敏度高、可用肉眼直接判定結果,為致病性副溶血弧菌的檢測提供一種簡單、快速、有效的方法。 目前,單克隆抗體技術廣泛應用在細菌檢測領域,并取得良好的效果。本文利用提取純化得到的TDH聯(lián)合佐劑多次免疫小鼠,根據雜交瘤原理,依次通過動物免疫、細胞融合、單克隆抗體的篩選和克隆,最后對雜交瘤細胞的親和性、抗體類型、特異性、穩(wěn)定性等性質進行了研究,成功研制出抗TDH鼠性單克隆抗體,實驗結果表明,本實驗制作的TDH單克隆抗體特異性高、穩(wěn)定性強、親和力高。 鑒于TDH和致病性副溶血弧菌感染的密切關系,利用TDH單克隆抗體初步探索應用在副溶血弧菌檢測方面的前景。表明利用單克隆抗體為基礎在致病性副溶血弧菌檢測方面將具有巨大的應用前景,為開發(fā)膠體金技術檢測副溶血弧菌奠定一定的基礎。
[Abstract]:In this paper, the thermostable direct hemolysin (TDH) of Vibrio parahaemolyticus Vp was extracted and purified. The related properties were analyzed. The mouse monoclonal antibody against TDH was successfully prepared by hybridoma technique, and its properties were identified. In this paper, vibrio parahaemolyticus was cultured in a culture medium containing certain nutrients, and crude protein was extracted by salting out, using the characteristic of exocrine secretion of heat-resistant hemolytic toxin by Vibrio parahaemolyticus (Vibrio parahaemolyticus). The thermostable hemolytic toxin of Vibrio parahaemolyticus was purified by ion exchange chromatography and gel chromatography, and the main pathogenic factor of Vibrio parahaemolyticus TDH was obtained. Because we should use TDH to immunize animals and explore the immunological method for detection of Vibrio parahaemolyticus, several physicochemical properties such as denaturation temperature, toxicity and heat resistance were analyzed and identified. The results showed that the direct heat-resistant hemolytic toxin of Vibrio parahaemolyticus was a typical protein, and the toxin had a strong toxic effect, and a small amount of TDH intravenously injected mice could die in a short time. The heat resistance of TDH was analyzed by means of differential heat scanning and heating treatment, and it was proved that the toxin had strong heat resistance. In the experiment, we used TDH as immunogen to immunize mice successfully to obtain polyclonal immune serum, and detect pathogenic Vibrio parahaemolyticus according to Western dot blotting principle, and achieved good results. The optimal working concentration of specific immune serum and its sensitivity to pathogenic Vibrio parahaemolyticus and non-pathogenic Vibrio parahaemolyticus were studied. The results showed that this method could be used to detect pathogenic Vibrio parahaemolyticus secreting TDH. It is insensitive to non-pathogenic Vibrio parahaemolyticus, simple in operation, high in sensitivity, and can be directly determined by naked eye, which provides a simple, rapid and effective method for the detection of pathogenic Vibrio parahaemolyticus. At present, monoclonal antibody technology is widely used in the field of bacterial detection, and achieved good results. According to the principle of hybridoma, the mice were immunized with TDH combined with adjuvant for several times. According to the principle of hybridoma, the mice were immunized by animal immunity, cell fusion, screening and cloning of monoclonal antibodies. Finally, the affinity and antibody type of hybridoma cells were obtained. The specificity, stability and other properties of the monoclonal antibody against TDH mouse were successfully developed. The experimental results showed that the TDH monoclonal antibody prepared in this experiment had high specificity, strong stability and high affinity. In view of the close relationship between TDH and pathogenic vibrio parahaemolyticus infection, the application of TDH monoclonal antibody in the detection of Vibrio parahaemolyticus was preliminarily explored. The results showed that the application of monoclonal antibody in the detection of pathogenic Vibrio parahaemolyticus would have a great prospect and lay a foundation for the development of colloidal gold technology for the detection of Vibrio parahaemolyticus.
【學位授予單位】：上海海洋大學
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：R392.1

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