副溶血弧菌耐熱溶血毒素單克隆抗體的研究
本文選題:副溶血弧菌 + 耐熱溶血毒素 ; 參考:《上海海洋大學(xué)》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(duì)其相關(guān)性質(zhì)進(jìn)行了分析,通過雜交瘤技術(shù)成功制備出TDH的鼠性單克隆抗體,并對(duì)其性質(zhì)進(jìn)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(yíng)養(yǎng)成分的細(xì)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(jīng)過離子交換層析及凝膠多次層析方法對(duì)副溶血弧菌耐熱溶血毒素進(jìn)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(dòng)物,研究探索免疫學(xué)方法檢測(cè)副溶血弧菌方法,接下來(lái)從變性溫度、毒性、耐熱性等幾個(gè)理化相關(guān)性質(zhì)進(jìn)了分析和鑒定。結(jié)果表明,副溶血弧菌直接耐熱溶血毒素是典型的蛋白質(zhì);此毒素具有較強(qiáng)的毒性效應(yīng),少量TDH靜脈注射小鼠就能是小鼠在短時(shí)間內(nèi)死亡;同時(shí),本研究還利用了差示熱量掃描法和加熱處理方法對(duì)TDH的耐熱性進(jìn)行了分析,證明了此毒素具有較強(qiáng)的耐熱性。 實(shí)驗(yàn)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(jù)Western斑點(diǎn)印跡原理檢測(cè)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(duì)致病性副溶血弧菌與非致病性副溶血弧菌的敏感性,結(jié)果表明此方法可針對(duì)性的檢測(cè)分泌TDH的致病性副溶血弧菌,對(duì)非致病性副溶血弧菌不敏感,操作簡(jiǎn)單、靈敏度高、可用肉眼直接判定結(jié)果,為致病性副溶血弧菌的檢測(cè)提供一種簡(jiǎn)單、快速、有效的方法。 目前,單克隆抗體技術(shù)廣泛應(yīng)用在細(xì)菌檢測(cè)領(lǐng)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(jù)雜交瘤原理,依次通過動(dòng)物免疫、細(xì)胞融合、單克隆抗體的篩選和克隆,最后對(duì)雜交瘤細(xì)胞的親和性、抗體類型、特異性、穩(wěn)定性等性質(zhì)進(jìn)行了研究,成功研制出抗TDH鼠性單克隆抗體,實(shí)驗(yàn)結(jié)果表明,本實(shí)驗(yàn)制作的TDH單克隆抗體特異性高、穩(wěn)定性強(qiáng)、親和力高。 鑒于TDH和致病性副溶血弧菌感染的密切關(guān)系,利用TDH單克隆抗體初步探索應(yīng)用在副溶血弧菌檢測(cè)方面的前景。表明利用單克隆抗體為基礎(chǔ)在致病性副溶血弧菌檢測(cè)方面將具有巨大的應(yīng)用前景,為開發(fā)膠體金技術(shù)檢測(cè)副溶血弧菌奠定一定的基礎(chǔ)。
[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(xué)位授予單位】:上海海洋大學(xué)
【學(xué)位級(jí)別】:碩士
【學(xué)位授予年份】:2011
【分類號(hào)】:R392.1
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