副溶血弧菌耐热溶血毒素单克隆抗体的研究

发布时间：2018-05-09 06:53

本文选题：副溶血弧菌 + 耐热溶血毒素　；参考：《上海海洋大学》2011年硕士论文

【摘要】：本文提取与纯化副溶血弧菌(Vibrio parahaemolyticus,Vp)耐热溶血毒素(thermostable direct hemolysin,TDH),对其相关性质进行了分析,通过杂交瘤技术成功制备出TDH的鼠性单克隆抗体,并对其性质进行了鉴定。本文利用副溶血弧菌胞外分泌耐热溶血毒素这一特性,在含有一定营养成分的细菌培养液增殖培养致病性副溶血弧菌,盐析提取粗蛋白,之后经过离子交换层析及凝胶多次层析方法对副溶血弧菌耐热溶血毒素进行了纯化,最终纯化得到了副溶血弧菌主要致病因子TDH。由于下面要利用TDH免疫动物,研究探索免疫学方法检测副溶血弧菌方法,接下来从变性温度、毒性、耐热性等几个理化相关性质进了分析和鉴定。结果表明,副溶血弧菌直接耐热溶血毒素是典型的蛋白质;此毒素具有较强的毒性效应,少量TDH静脉注射小鼠就能是小鼠在短时间内死亡;同时,本研究还利用了差示热量扫描法和加热处理方法对TDH的耐热性进行了分析,证明了此毒素具有较强的耐热性。实验中,我们将TDH作为免疫原免疫小鼠成功得到多克隆免疫血清,根据Western斑点印迹原理检测致病性副溶血弧菌,取得了良好的效果。探讨了特异性免疫血清的最佳工作浓度以及分别对致病性副溶血弧菌与非致病性副溶血弧菌的敏感性,结果表明此方法可针对性的检测分泌TDH的致病性副溶血弧菌,对非致病性副溶血弧菌不敏感,操作简单、灵敏度高、可用肉眼直接判定结果,为致病性副溶血弧菌的检测提供一种简单、快速、有效的方法。目前,单克隆抗体技术广泛应用在细菌检测领域,并取得良好的效果。本文利用提取纯化得到的TDH联合佐剂多次免疫小鼠,根据杂交瘤原理,依次通过动物免疫、细胞融合、单克隆抗体的筛选和克隆,最后对杂交瘤细胞的亲和性、抗体类型、特异性、稳定性等性质进行了研究,成功研制出抗TDH鼠性单克隆抗体,实验结果表明,本实验制作的TDH单克隆抗体特异性高、稳定性强、亲和力高。鉴于TDH和致病性副溶血弧菌感染的密切关系,利用TDH单克隆抗体初步探索应用在副溶血弧菌检测方面的前景。表明利用单克隆抗体为基础在致病性副溶血弧菌检测方面将具有巨大的应用前景,为开发胶体金技术检测副溶血弧菌奠定一定的基础。
[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.
【学位授予单位】：上海海洋大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392.1

【参考文献】