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

【摘要】：志賀氏菌又稱痢疾桿菌,是引發(fā)我國(guó)感染性腹瀉、嚴(yán)重危害人類健康的食源性致病菌。它主要侵害結(jié)腸,引發(fā)潰瘍、粘液性血性腹瀉,對(duì)嬰幼兒有較高的感染率和死亡率。加強(qiáng)對(duì)志賀氏菌的監(jiān)測(cè),有助于預(yù)防和控制大規(guī)模感染疫情的爆發(fā)。目前,針對(duì)食源性致病菌的檢測(cè)方法主要有傳統(tǒng)的生化培養(yǎng)、PCR檢測(cè)和免疫學(xué)方法等。免疫學(xué)方法因其簡(jiǎn)單、快速、成本低廉等優(yōu)點(diǎn)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(jiǎn)便篩查志賀氏菌的膠體金免疫試紙條檢測(cè)方法,用于其臨床診斷及食品衛(wèi)生監(jiān)測(cè)。 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(jià)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(yīng),獲得了抗志賀氏菌的多克隆抗體。通過(guò)細(xì)胞融合技術(shù),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(xì)胞株。采用辛酸硫酸銨沉淀法對(duì)單克隆細(xì)胞株4G9的腹水進(jìn)行純化,獲得單克隆抗體,可與純化多抗配對(duì)使用。用膠體金標(biāo)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(cè)線和質(zhì)控線,制備了志賀氏菌的膠體金免疫層析試紙條。檢測(cè)志賀氏菌純培養(yǎng)物的靈敏度為105CFU/mL:在添加108CFU/mL的長(zhǎng)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(xì)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(duì)發(fā)現(xiàn)存在終止密碼子,因此只把VH分別構(gòu)建到pET-lic、pGEX-4T-1載體中,轉(zhuǎn)入大腸桿菌中表達(dá),通過(guò)ELISA方法驗(yàn)證了兩種表達(dá)產(chǎn)物均能與志賀氏菌結(jié)合,但親和力較低,不適合用于制備檢測(cè)志賀氏菌的免疫層析試紙條。 本文研究結(jié)果將為建立其它食源性致病菌的快速檢測(cè)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(xué)位授予單位】：南昌大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2013
【分類號(hào)】：R392

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 楊介鉆,張眾,馬驪,姚新生,周明乾,王祥斌,王小寧;抗人卵巢癌/抗人CD3單鏈雙特異性抗體的生物學(xué)活性研究[J];癌癥;2005年07期

2 白麗,錢金h?,王晶;應(yīng)用辛酸硫酸胺法提取小鼠腹水和血清中的IgG抗體[J];大理醫(yī)學(xué)院學(xué)報(bào);2000年04期

3 張長(zhǎng)弓,金顏輝,黃印堯,方瑩,梁全順;口蹄疫抗體免疫金標(biāo)快速檢測(cè)試紙法的建立[J];福建畜牧獸醫(yī);2004年01期

4 邱楊;唐麗霞;趙麗;劉建麗;;志賀氏菌實(shí)時(shí)熒光PCR檢測(cè)試劑的研制[J];現(xiàn)代食品科技;2008年11期

5 黃洋,趙暉,招為國(guó),張宗顯;SPA方法快速檢驗(yàn)食品志賀氏菌的研究[J];湖北化工;1999年03期

6 李君文,晁福寰,史秀全,王新為,鄭金來(lái),宋農(nóng),靳連群,金敏;志賀氏菌的RAPD鑒定研究[J];解放軍預(yù)防醫(yī)學(xué)雜志;2003年01期

7 張靖;張富昌;;基因工程抗體與制備技術(shù)的研究及應(yīng)用[J];科技情報(bào)開(kāi)發(fā)與經(jīng)濟(jì);2006年10期

8 宮航宇;石銘;韓博;;基因探針技術(shù)在傳染病診斷中的應(yīng)用[J];臨床肝膽病雜志;2005年06期

9 張振奇;孫忠偉;王利;;益生菌在嬰幼兒配方奶粉中應(yīng)用的研究[J];中國(guó)乳業(yè);2006年11期

10 熊友華;高愛(ài)中;任東明;丁旭貝;王磊;;羅丹明B單克隆抗體的的制備及鑒定[J];免疫學(xué)雜志;2011年04期

，

本文編號(hào)：1782846