志贺氏菌多克

发布时间：2018-04-21 14:36

本文选题：志贺氏菌 + 多克隆抗体　；参考：《南昌大学》2013年硕士论文

【摘要】：志贺氏菌又称痢疾杆菌,是引发我国感染性腹泻、严重危害人类健康的食源性致病菌。它主要侵害结肠,引发溃疡、粘液性血性腹泻,对婴幼儿有较高的感染率和死亡率。加强对志贺氏菌的监测,有助于预防和控制大规模感染疫情的爆发。目前,针对食源性致病菌的检测方法主要有传统的生化培养、PCR检测和免疫学方法等。免疫学方法因其简单、快速、成本低廉等优点而备受青睐。本文旨在制备多种抗志贺氏菌的抗体,建立一种快速、简便筛查志贺氏菌的胶体金免疫试纸条检测方法,用于其临床诊断及食品卫生监测。采用志贺氏菌混合菌体碎片作为免疫原免疫家兔,制备获得了效价为1：1280000的抗血清。经菌体逆向吸附法和辛酸硫酸铵沉淀法纯化后,有效去除了与大肠杆菌和阪崎肠杆菌等杂菌的交叉反应,获得了抗志贺氏菌的多克隆抗体。通过细胞融合技术,筛选得到4株能稳定分泌抗志贺氏菌单克隆抗体的杂交瘤细胞株。采用辛酸硫酸铵沉淀法对单克隆细胞株4G9的腹水进行纯化,获得单克隆抗体,可与纯化多抗配对使用。用胶体金标记单克隆抗体,将多克隆抗体和驴抗鼠IgG分别喷涂于硝酸纤维素膜作为检测线和质控线,制备了志贺氏菌的胶体金免疫层析试纸条。检测志贺氏菌纯培养物的灵敏度为105CFU/mL:在添加108CFU/mL的长双歧杆菌和阪崎肠杆菌的干扰下,其牛奶样品中的志贺氏菌的灵敏度为106CFU/mL.本文从制备的志贺氏菌单抗细胞株4G9中获得了VL、VH基因的序列,发现其分别属于κ链和γ链。由于VL经比对发现存在终止密码子,因此只把VH分别构建到pET-lic、pGEX-4T-1载体中,转入大肠杆菌中表达,通过ELISA方法验证了两种表达产物均能与志贺氏菌结合,但亲和力较低,不适合用于制备检测志贺氏菌的免疫层析试纸条。本文研究结果将为建立其它食源性致病菌的快速检测方法,提供可借鉴的研究模式。
[Abstract]:Shigella, also known as Shigella dysenteriae, is a foodborne pathogen that causes infectious diarrhea and seriously endangers human health. It mainly invades the colon, causes ulcers, mucous bloody diarrhea, and has a high infection rate and mortality in infants. Strengthening the surveillance of Shigella can help to prevent and control the outbreak of large-scale infection. At present, traditional PCR and immunological methods are mainly used for the detection of foodborne pathogenic bacteria. Immunological methods are popular for their simplicity, rapidity and low cost. The purpose of this paper is to prepare a variety of antibodies against Shigella and to establish a rapid and simple method for detecting Shigella by colloidal gold immunoassay for clinical diagnosis and food hygiene monitoring. The antiserum of 1: 1280000 was prepared by immunizing rabbits with Shigella mixed bacterial fragments. The cross reaction with Escherichia coli and Enterobacter sakazakii was effectively removed and polyclonal antibodies against Shigella spp. Four hybridoma cell lines which secreted monoclonal antibodies against Shigella stably were screened by cell fusion technique. The ascites of monoclonal cell line 4G9 were purified by ammonium octanoate precipitation method, and the monoclonal antibody was obtained, which can be paired with the purified polyclonal antibody. Colloidal gold immunochromatographic test strip of Shigella was prepared by using colloidal gold labeled monoclonal antibody and donkey anti-mouse IgG sprayed on nitrocellulose membrane as detection line and quality control line respectively. The sensitivity of the pure culture of Shigella was 105 CFU / mL: the sensitivity of Shigella in milk samples was 106 CFU / mL under the interference of Bifidobacterium longus and Enterobacter sakazakii added with 108CFU/mL. In this paper, we obtained the sequence of VLVH gene from Shigella spp McAb cell line 4G9, and found that it belongs to 魏 chain and 纬 chain, respectively. Because of the existence of termination codon in VL, VH was only constructed into pET-liche pGEX-4T-1 vector and expressed in Escherichia coli. The ELISA method showed that both of the two expressed products could bind to Shigella, but their affinity was low. It is not suitable for the preparation of immunochromatographic test strips for Shigella. The results of this study will provide a model for the rapid detection of other foodborne pathogens.
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R392

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