大肠杆菌O157的PCR检测及Vero毒素单克隆抗体的制备

发布时间：2018-05-27 10:19

  本文选题：大肠杆菌O157 + 毒力基因　； 参考：《南京农业大学》2006年硕士论文

【摘要】：大肠杆菌O157为食源性病原菌，可引起严重的食物中毒，及致人死亡，建立特异快速的检测方法是目前控制食品安全的关键。大多数大肠杆菌O157产Vero毒素(VT)，已知VT是大肠杆菌O157感染引起溶血性尿毒综合征的主要毒力因子之一，检测VT可以直接判定样本是否具有致病性毒素。本课题建立了大肠杆菌O157快速灵敏的基因检测方法和制备了VT的单克隆抗体，，两者结合应用既可以检测大肠杆菌O157的毒力基因又可以检测基因产物，可根据不同条件选择不同的检测方法以提高样本的检测率。 根据GenBank中编码大肠杆菌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(紧密素基因)5种基因的特异性序列，设计合成5对引物，建立了快速鉴定大肠杆菌O157的多重PCR方法。该方法的检测敏感度为细菌纯培养物为10~4CFU／ml，鸡肉组织样品含菌3-4CFU／g经预增菌8h后能检出。用此方法检测2000-2004年间临床分离的64株动物组织和粪便的菌株，结果10株鉴定为大肠杆菌O157，其中8株能扩增上述5条特异性条带，与预期产物完全一致，2株能扩增出除vt1基因外的4条特异性条带，其他54株菌株只能扩增出大肠杆菌16s rRNA，与血清凝集试验结果完全吻合。该方法通过预增菌能检测动物源性食品中的大肠杆菌O157，且能从基因水平直接鉴定该菌是否产VT，特异性强，为检测和鉴定大肠杆菌O157提供了一个新方法。 以大肠杆菌O157 DNA为模板扩增VT2-B亚单位，纯化后经EcoR Ⅰ和Xho Ⅰ酶切，导入带有谷胱甘肽S转移酶(GST)的融合表达载体pGEX_(4T-2)，构建重组表达质粒pGEX_(4T-2)-VT2-B。转化到大肠杆菌BL21中，经IPTG诱导，实现了VT2-B-GST融合蛋白的高表达，表达量约占菌体总蛋白的35％，为可溶性蛋白。经GST亲和层析柱纯化，得到纯度较高的VT2-B-GST融合蛋白。 用纯化的VT2-B-GST融合蛋白免疫BALB／C小鼠，取脾细胞与骨髓瘤细胞Sp2／0融合，用间接ELISA筛选融合蛋白的阳性克隆，剔除与细胞壁杂蛋白交叉反应的阳性孔，再用有限稀释法获得单克隆，最后获得2株针对VT2-B亚单位的单克隆抗体细胞株：4B×F10×D4和4B×8G×11F。将10~5-10~7个4B×8G×11F细胞注射到BALB／C小鼠腹腔，制备腹水，ELISA效价为10~4。用Western blot鉴定此单抗，结果与融合蛋白反应呈阳性，与GST蛋白反应呈阴性，证明此单抗为抗VT2-B亚单位的特异性单
[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

本文编号：1941620