兰州大学机构库 >生命科学学院
肠炎沙门氏菌LPS单克隆抗体的制备及初步应用
Alternative TitlePreparation and application of monoclonal antibodies of lipopolysaccharide of Salmonella enteritidies
鲁学萍
Thesis Advisor李克生
2011-05-17
Degree Grantor兰州大学
Place of Conferral兰州
Degree Name硕士
Keyword肠炎沙门氏菌 脂多糖(LPS) 单克隆抗体
Abstract肠炎沙门氏菌是一种常见的人兽共患的肠道致病菌,常常引起人类和禽畜的肠胃炎和败血症,同时还可以造成人类食物中毒,从而带来巨大的经济损失。因此,对该菌的检测一直是食品安全检测的重点,建立一种快速、准确、灵敏的检测方法是预防该菌食物中毒的主要措施。细菌的检测方法种类繁多,其中免疫学检测方法(尤其是依赖于单抗和多抗的ELISA检测方法),由于具有操作简单、灵敏度高、特异性强等优点,被广泛用于食品中该菌的检测。单克隆抗体由于特异性好,在细菌的分类、鉴定、检测以及相关抗原的纯化、分离和鉴定中都有重要的意义。 LPS是位于革兰氏阴性菌细胞表面的主要抗原,它由核心多糖、O-特异性侧链多糖和类脂A三部分组成,其多糖部分决定了菌体的种属特异性。本实验分别采用热酚水法与水饱和冷酚法抽提肠炎沙门氏菌的LPS抗原,并对其生物学特性进行了初步的研究。结果显示,热酚水法与水饱和冷酚法均可制备活性较好的肠炎沙门氏菌LPS,但冷酚法更加简单、方便、并且提取的样品纯度高。SDS-PAGE电泳银染后,LPS呈现明显的梯度型条带。间接ELISA法检测制备的LPS与常见肠道致病菌多价和单价抗血清的交叉性,结果表明制备的LPS抗原特异性较好,可进一步用于单克隆抗体的制备。 以65℃、30 min灭活的肠炎沙门氏菌免疫BALB/c小鼠,经三次免疫后,取其脾细胞与小鼠骨髓瘤SP2/0细胞进行融合。以纯化的LPS抗原作为筛选抗原,间接ELISA方法筛选能分泌高特异抗体的阳性杂交瘤细胞株。采用间接ELISA方法和western blot测定其效价、亚类和特异性。选取效价高、特异性好的单克隆抗体株进行HRP标记,初步建立双抗体夹心ELISA。结果显示,两次融合筛选共得到9株能稳定分泌抗肠炎沙门氏菌LPS抗原的单克隆抗体细胞株。间接ELISA检测表明单抗1D3、2E5、6D7和7E2四株细胞效价比较高、特异性较强。选抗原位点配对好的mAb6D7作为捕获抗体,mAb2E5作为检测抗体初步建立了双抗体夹心ELISA方法。该检测体系特异性好,对肠炎沙门氏菌的最低检测限达4.0×105 cfu/ml。制备的抗肠炎沙门氏菌的单克隆抗体特异性好,可进一步用于肠炎沙门氏菌LPS结构和生物学特性的研究。
Other AbstractSalmonella enteritidis is a common enteric pathogenic bacteria for both human being and animal, they can result in human and anminal disease and caused food poisoning in human beings, which usually result in great economic losses. It is necessary to establish a rapid, accurate and sensitive detected method for reducing the outbreaks of humen infections. There are several method can be used to bacterial detection of in the food, serological techniques (especially the ELISA assay employing polyclonal or monoclonal antibodies) is more sensitive and simpler for detection of salmonella enteritidis in the food. Although polyclonal antibodies have been successfully used to detect and classify Salmonella, they are not efficient in detecting minor antigenic variations. Monoclonal antibodies have great potential for identifying, isolating and purifying bacterial antigens and also in defineing the role of specific antigens in diagnosis, classification and immunity to infection. Lipopolysaccharide (LPS) is a major surface component of gram-negative bacterial cell envelopes. Most types of LPS are composed of three distinct regions: O antigenic polysaccharide chains, core oligosaccharide and the lipid A. O antigenic polysaccharide structure confers serotype specificity on a species or strain of bacteria. We extracted the LPS antigen of Salmonella enteritidis strain both by hot phenol water and cold phenol method, and studied the biologic characteristic of LPS antigen. The results showed that cold water-saturation phenol extract method is more convinient and rapider than hot phenol water. Following SDS-PAGE, polyacrylamide gels are silver stained to visualize the LPS banding patterns. According to the indirect ELISA, there is no cross-reaction with most antisera of other enteric bacterias. These results show that the extracted SE-LPS antigen has hightly specificity and can be used for the monoclonal antibodies studies. The BALB/c were immunized with 65℃, 30min heat killed Salmonella enteritidis. The spleen of BALB/c mice immunized were harvested and fused with SP2/0 cell. Specific hybridoma clones were selected after testing culture supernatants by ELISA. Nine clony mAbs were obtained by two times fusions. These mAbs’titers, specificcites and Ig subclasses were determined by western blot and indirect ELISA. We established a sandwich enzyme-linked immunosorbent assay (S-ELISA) for detecting Salmonella enteritidis using mAb 6D7 as a detecting antibody and mAb 2E5...
URL查看原文
Language中文
Document Type学位论文
Identifierhttps://ir.lzu.edu.cn/handle/262010/221637
Collection生命科学学院
Recommended Citation
GB/T 7714
鲁学萍. 肠炎沙门氏菌LPS单克隆抗体的制备及初步应用[D]. 兰州. 兰州大学,2011.
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