本文選題：志賀氏菌 + 多克隆抗體　； 參考：《南昌大學》2013年碩士論文

【摘要】：志賀氏菌又稱痢疾桿菌,是引發(fā)我國感染性腹瀉、嚴重危害人類健康的食源性致病菌。它主要侵害結腸,引發(fā)潰瘍、粘液性血性腹瀉,對嬰幼兒有較高的感染率和死亡率。加強對志賀氏菌的監(jiān)測,有助于預防和控制大規(guī)模感染疫情的爆發(fā)。目前,針對食源性致病菌的檢測方法主要有傳統(tǒng)的生化培養(yǎng)、PCR檢測和免疫學方法等。免疫學方法因其簡單、快速、成本低廉等優(yōu)點而備受青睞。本文旨在制備多種抗志賀氏菌的抗體,建立一種快速、簡便篩查志賀氏菌的膠體金免疫試紙條檢測方法,用于其臨床診斷及食品衛(wèi)生監(jiān)測。 采用志賀氏菌混合菌體碎片作為免疫原免疫家兔,制備獲得了效價為1：1280000的抗血清。經(jīng)菌體逆向吸附法和辛酸硫酸銨沉淀法純化后,有效去除了與大腸桿菌和阪崎腸桿菌等雜菌的交叉反應,獲得了抗志賀氏菌的多克隆抗體。通過細胞融合技術,篩選得到4株能穩(wěn)定分泌抗志賀氏菌單克隆抗體的雜交瘤細胞株。采用辛酸硫酸銨沉淀法對單克隆細胞株4G9的腹水進行純化,獲得單克隆抗體,可與純化多抗配對使用。用膠體金標記單克隆抗體,將多克隆抗體和驢抗鼠IgG分別噴涂于硝酸纖維素膜作為檢測線和質控線,制備了志賀氏菌的膠體金免疫層析試紙條。檢測志賀氏菌純培養(yǎng)物的靈敏度為105CFU/mL:在添加108CFU/mL的長雙歧桿菌和阪崎腸桿菌的干擾下,其牛奶樣品中的志賀氏菌的靈敏度為106CFU/mL.本文從制備的志賀氏菌單抗細胞株4G9中獲得了VL、VH基因的序列,發(fā)現(xiàn)其分別屬于κ鏈和γ鏈。由于VL經(jīng)比對發(fā)現(xiàn)存在終止密碼子,因此只把VH分別構建到pET-lic、pGEX-4T-1載體中,轉入大腸桿菌中表達,通過ELISA方法驗證了兩種表達產(chǎn)物均能與志賀氏菌結合,但親和力較低,不適合用于制備檢測志賀氏菌的免疫層析試紙條。 本文研究結果將為建立其它食源性致病菌的快速檢測方法,提供可借鑒的研究模式。
[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 緯 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.
【學位授予單位】：南昌大學
【學位級別】：碩士
【學位授予年份】：2013
【分類號】：R392

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