本文選題：空腸彎曲桿菌　切入點(diǎn)：CadF蛋白　出處：《東北農(nóng)業(yè)大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：空腸彎曲桿菌(Campylobacter jejuni,C.jejuni),是一種食源性致病菌,近年來,由其引發(fā)的食品安全問題日益突出。全世界每年有大約4-5億人感染該菌,主要臨床表現(xiàn)是以嘔吐、腹瀉癥狀的空腸彎曲桿菌腸炎,甚至繼發(fā)格林-巴利綜合征(Guillain-Barri syndrome,GBS)。開展流行病學(xué)調(diào)查并對(duì)該病進(jìn)行實(shí)時(shí)監(jiān)測(cè)是有效控制該菌感染的根本途徑,一種快速、準(zhǔn)確、敏感性強(qiáng)、適應(yīng)性強(qiáng)的檢測(cè)方法亟需建立。本研究以C.jejuni Q11株為研究對(duì)象,DNAStar軟件進(jìn)行序列分析并設(shè)計(jì)引物,PCR方法擴(kuò)增目的基因cad F,然后利用p ET32a對(duì)cad F基因進(jìn)行原核表達(dá),構(gòu)建重組質(zhì)粒pET32a-Cad F,并將重組質(zhì)粒轉(zhuǎn)入大腸桿菌BL21(DE3)感受態(tài)細(xì)胞,在37℃條件下,以1mmol/L IPTG對(duì)重組蛋白進(jìn)行誘導(dǎo)表達(dá)。誘導(dǎo)表達(dá)后的重組蛋白通過SDS-PAGE及Western blot進(jìn)行驗(yàn)證。SDS-PAGE分析表明,重組蛋白pET32a-Cad F分子量大小為53 ku,以包涵體形式存在于沉淀中;Western blot分析表明,該蛋白可與空腸彎曲桿菌全菌陽性血清產(chǎn)生特異性反應(yīng),具有良好的反應(yīng)原性。采用親和層析的方法對(duì)帶有His-Tag標(biāo)簽的蛋白進(jìn)行純化,以復(fù)性的蛋白作為抗原建立了檢測(cè)空腸彎曲桿菌血清的間接ELISA方法。對(duì)間接ELISA方法的條件進(jìn)行優(yōu)化,采用Cut-Off值法確定間接ELISA的陰陽臨界值。最終確定所建立方法的最佳條件是:在37℃條件下,抗原包被濃度為1.05μg/m L;包被時(shí)間為2 h;封閉條件為5%脫脂乳封閉1 h;陽性血清稀釋倍數(shù)為1:100,作用時(shí)間為45 min;二抗作用濃度和時(shí)間分別為1:5000、45 min,顯色時(shí)間為10 min,臨界值為0.339。利用大腸桿菌、沙門氏菌、多殺性巴氏桿菌等常見雞病的陽性血清驗(yàn)證所建立檢測(cè)方法的敏感性、批內(nèi)重復(fù)性及批間重復(fù)性驗(yàn)證對(duì)該方法的可重復(fù)性進(jìn)行了解,結(jié)果表明此方法具有良好敏感性、重復(fù)性及特異性;對(duì)血清樣品檢測(cè)表明:該方法可應(yīng)用于臨床樣品的初步檢測(cè)。所建立間接ELISA方法不僅為空腸彎曲桿菌的流行病學(xué)調(diào)查提供了初步檢測(cè)方法,更為疾病的預(yù)防和控制提供有效依據(jù)。
[Abstract]:Campylobacter jejunianus, a foodborne pathogen, has been causing increasing food safety problems in recent years. About 400 million people worldwide are infected with the bacterium each year, the main clinical manifestation of which is vomiting. Campylobacter jejuni enteritis with diarrhea symptoms, and even Guillain-Barri syndromefus GBSN secondary to Guillain-Barri syndromefus syndrome. Epidemiological investigation and real-time monitoring of the disease are the fundamental ways to effectively control the infection of Campylobacter jejuni. It is a rapid, accurate and sensitive way to control the infection of Campylobacter jejuni. In this study, the sequence analysis of C. jejuni Q11 strain was carried out, and a primer PCR method was designed to amplify the target gene cad F, and then prokaryotic expression of cad F gene was carried out by p ET32a. The recombinant plasmid pET32a-Cad F was constructed, and the recombinant plasmid was transferred into Escherichia coli BL21 (DE3) competent cells. At 37 鈩，

本文編號(hào)：1571878