本文選題：大腸桿菌O157 + 毒力基因��； 參考：《南京農(nóng)業(yè)大學(xué)》2006年碩士論文

【摘要】：大腸桿菌O157為食源性病原菌，可引起嚴(yán)重的食物中毒，及致人死亡，建立特異快速的檢測方法是目前控制食品安全的關(guān)鍵。大多數(shù)大腸桿菌O157產(chǎn)Vero毒素(VT)，已知VT是大腸桿菌O157感染引起溶血性尿毒綜合征的主要毒力因子之一，檢測VT可以直接判定樣本是否具有致病性毒素。本課題建立了大腸桿菌O157快速靈敏的基因檢測方法和制備了VT的單克隆抗體，，兩者結(jié)合應(yīng)用既可以檢測大腸桿菌O157的毒力基因又可以檢測基因產(chǎn)物，可根據(jù)不同條件選擇不同的檢測方法以提高樣本的檢測率。 根據(jù)GenBank中編碼大腸桿菌16s rRNA的基因、rfbE(O157抗原基因)、vt1(Vero毒素1基因)、vt2(Vero毒素2基因)和eaeA(緊密素基因)5種基因的特異性序列，設(shè)計(jì)合成5對引物，建立了快速鑒定大腸桿菌O157的多重PCR方法。該方法的檢測敏感度為細(xì)菌純培養(yǎng)物為10~4CFU／ml，雞肉組織樣品含菌3-4CFU／g經(jīng)預(yù)增菌8h后能檢出。用此方法檢測2000-2004年間臨床分離的64株動(dòng)物組織和糞便的菌株，結(jié)果10株鑒定為大腸桿菌O157，其中8株能擴(kuò)增上述5條特異性條帶，與預(yù)期產(chǎn)物完全一致，2株能擴(kuò)增出除vt1基因外的4條特異性條帶，其他54株菌株只能擴(kuò)增出大腸桿菌16s rRNA，與血清凝集試驗(yàn)結(jié)果完全吻合。該方法通過預(yù)增菌能檢測動(dòng)物源性食品中的大腸桿菌O157，且能從基因水平直接鑒定該菌是否產(chǎn)VT，特異性強(qiáng)，為檢測和鑒定大腸桿菌O157提供了一個(gè)新方法。 以大腸桿菌O157 DNA為模板擴(kuò)增VT2-B亞單位，純化后經(jīng)EcoR Ⅰ和Xho Ⅰ酶切，導(dǎo)入帶有谷胱甘肽S轉(zhuǎn)移酶(GST)的融合表達(dá)載體pGEX_(4T-2)，構(gòu)建重組表達(dá)質(zhì)粒pGEX_(4T-2)-VT2-B。轉(zhuǎn)化到大腸桿菌BL21中，經(jīng)IPTG誘導(dǎo)，實(shí)現(xiàn)了VT2-B-GST融合蛋白的高表達(dá)，表達(dá)量約占菌體總蛋白的35％，為可溶性蛋白。經(jīng)GST親和層析柱純化，得到純度較高的VT2-B-GST融合蛋白。 用純化的VT2-B-GST融合蛋白免疫BALB／C小鼠，取脾細(xì)胞與骨髓瘤細(xì)胞Sp2／0融合，用間接ELISA篩選融合蛋白的陽性克隆，剔除與細(xì)胞壁雜蛋白交叉反應(yīng)的陽性孔，再用有限稀釋法獲得單克隆，最后獲得2株針對VT2-B亞單位的單克隆抗體細(xì)胞株：4B×F10×D4和4B×8G×11F。將10~5-10~7個(gè)4B×8G×11F細(xì)胞注射到BALB／C小鼠腹腔，制備腹水，ELISA效價(jià)為10~4。用Western blot鑒定此單抗，結(jié)果與融合蛋白反應(yīng)呈陽性，與GST蛋白反應(yīng)呈陰性，證明此單抗為抗VT2-B亞單位的特異性單
[Abstract]:Escherichia coli O157 is a foodborne pathogen which can cause severe food poisoning and death. It is the key to control food safety to establish a specific and rapid method for detection of Escherichia coli O157. Most of E. coli O157 produce Vero toxin, VT is known to be one of the main virulence factors of hemolytic uremic syndrome caused by E. coli O157 infection. The detection of VT can directly determine whether the samples have pathogenic toxin. In this paper, a rapid and sensitive gene detection method for E. coli O157 and a monoclonal antibody to VT were developed. The combination of the two methods can be used to detect the virulence gene of E. coli O157 as well as the gene product. Different detection methods can be selected according to different conditions to improve the detection rate of samples. Five pairs of primers were designed and synthesized according to the specific sequences of the genes encoding 16 s rRNA of E. coli in GenBank, namely the gene of Vt1, Vero toxin 1 and the gene of Vero virotoxin 2, and eaeA (5 genes of close-in gene), and the gene encoding E. coli 16s rRNA was used to design and synthesize 5 pairs of primers. A multiplex PCR method for rapid identification of Escherichia coli O157 was established. The sensitivity of this method was 10 ~ 4 CFU / ml, and the 3-4CFU/g of chicken tissue could be detected after 8 hours of inoculation. This method was used to detect 64 strains of animal tissues and feces isolated from 2000 to 2004. The results showed that 10 strains were identified as E. coli O157, 8 of which could amplify the above 5 specific bands. Two strains could amplify four specific bands except vt1 gene, while the other 54 strains could only amplify 16s rRNAs of Escherichia coli, which was consistent with the results of serum agglutination test. This method can detect Escherichia coli O157 in animal-derived food by pre-inoculation, and can directly identify whether the bacterium produces VT from gene level, which provides a new method for detection and identification of Escherichia coli O157. E. coli O157 DNA was used as template to amplify the VT2-B subunit. After purification, EcoR 鈪

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