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1、上海交通大学硕士学位论文免疫胶体金技术快速检测大肠杆菌O157和志贺毒素姓名：高成秀申请学位级别：硕士专业：预防兽医学指导教师：严亚贤;孙建和20080101上海交大硕士论文免疫胶体金技术快速检测大肠杆菌 O157和志贺毒素摘要摘要大肠杆菌O157是产志贺毒素大肠杆菌（Shiga-producing Escherichia coli, STEC）的主要血清型，可以引起人和动物的腹泻、出血性结肠炎、溶血性尿毒综合征、血栓性血小板减少性紫癜。志贺毒素（Shiga toxin，Stxs）是大肠杆菌 O157的主要毒力因子之一，有两个生物型：Stx1和Stx2，只产生Stx2的菌株毒力比只分泌Stx1
2、和两者同时分泌的菌株毒力都强。Stx2致病剂量极低，且分泌胞外，Stx2的检测将直接有助于对分离菌株致病力强弱的判定，提高对高致病性大肠杆菌O157菌株的检出。因此本课题研究大肠杆菌O157和Stx2的胶体金层析法 (，)，进行高致病性大肠杆菌O157菌株检测和鉴定，以便更好的预防和控制大肠杆菌O157所致的人和动物的疾病。将本实验室构建的重组Stx2B亚单位的质粒pGEX-Stx2B转化入大肠杆菌BL21，然后采用PCR和酶切进行鉴定，将阳性转化菌进行重组蛋白的表达、鉴定、纯化，用纯化的融合蛋白多次免疫新西兰大白兔制备抗重组Stx2B的多克隆抗体。同时用大肠杆菌O157 ATCC43889全
3、菌体抗原多次免疫新西兰大白兔，制备抗大肠杆菌O157多克隆抗体。用硫酸胺法、辛酸-硫酸胺法、Protein G亲和层析3种方法分别纯化制备的多克隆抗体，结果表明：Protein G法纯化的抗重组Stx2B IgG纯度较其前两者纯，其金标多抗的稳定性较好，辛酸-硫酸胺法纯化的抗大肠杆菌O157 IgG的活性较Protein G纯化的好，其金标多抗的稳定性较好，因此本试验采用Protein G法纯化抗Stx2B血清，用辛酸-硫酸胺法纯化抗大肠杆菌O157血清。根据试纸条的侧向流动和胶体金的示踪功能，结合免疫学原理，分别建立并优化了检测大肠杆菌O157和Stx2的双抗体夹心免疫胶体金方法。测定了胶体
4、金颗粒的最优化反应体系：胶体金颗粒的大小为20nm；抗体与胶体金溶液结合的最佳pH约为8.2；最佳蛋白结合量分别为抗大肠杆菌O157 IgG为57g/mL，抗重组Stx2B IgG为III上海交大硕士论文摘要60g/mL；最佳稳定剂为 BSA；最佳缓冲液为pH8.2硼酸溶液；最佳金标保存液和洗涤液为5mM NaCl-1%BSA的pH 8.2的硼酸缓冲液；NC膜的封闭液为 3BSA的0.01mol/L PBST；Stx2试纸条和大肠杆菌O157试纸条的质控线（ C线）上的羊抗兔IgG的多克隆抗体最佳点样量均为1 g，其检测线（ T线）的抗Stx2的单克隆抗体的点样量为0.1g、抗大肠杆菌O157
5、的单克隆抗体的点样量为1g，结合垫上的金标抗大肠杆菌O157和重组Stx2B多克隆抗体点样量均为3g。用已建立的大肠杆菌O157免疫层析法对实验室保存的58株已鉴定的菌株进行检测，与血清凝集实验结果相比较，其阳性检出率的符合率达87.5%，检测下限为105CFU/mL，显色时间为35分钟，阳性检出率的批内平均变异系数为2.1%，阳性检出率的批间平均变异系数为6.9%。用已建立的Stx2免疫层析法对实验室保存的58株已鉴定的菌株进行检测，与 Vero细胞毒性实验相比较，其阳性检出率的符合率为50%，显色时间为510分钟，阳性检出率的批内平均变异系数为2.6%，阳性检出率的批间平均变异系数为28.
6、85%。大肠杆菌O157试纸条的特异性、重复性和敏感性较好，大肠杆菌O157免疫胶体金试纸条转化为商用试纸条是可行的。Stx2免疫胶体金试纸条在抗体效价、敏感性方面还需进一步优化，以提高阳性检出率和重复性。关键词：大肠杆菌O157；Stx2；免疫胶体金层析法；抗体纯化；反应体系；检测IV上海交大硕士论文ABSTRCTRAPID GOLD IMMUNOCHROMATOGRAPHY ASSAYSFOR DETECTION OFE.COLI O157 AND SHIGA TOXINABSTRACTShiga-producing Escherichia coli, especially serotyp
7、e O157, have been associatedwith hemorrhagic colitis, hemolytic uremic syndrome (HUS), and thromboticthrombocytopenic purpura. Stxs（Shiga toxin, Stxs）are the major pathoge nicity factors ofE.coli O157 which have been implicated in disease. The members of the Stx family ofE.coli consist of Stx1, Stx2
8、 and variants of Stx2, however, Stx2 is mostly greater thanStx1 and both of them which secreted by the same E.coli O157 in virulence. Its essentialto develop a method to detect directly Stx2. Stx2 has a low pathogenicity dose, andsecreted in the culture solution, therefore, detection for Stx2 associ
9、ated with E.coli O157was helpful to identify the high pathogenic E.coli O157 isolates. In this study, we haddeveloped sandwich GICA (Gold Immunochromatography Assay) of E.coli O157 andStx2 to diagnosis the high pathogenic E.coli O157 and Shiga toxin, then we would takenecesary steps to control and c
10、ure the desease infected by high pathogenic E.coli O157 inoutbreak situations.The recombinant expression vector designated as pGEX-Stx2B constructed by the labwas transformed into E.coli BL21 compotent cells, then the positive bacterium colonycontaining recombinant vector pGEX-Stx2 B identified by P
11、CR and digestion of theV上海交大硕士论文ABSTRCTendonucleases EcoR and Xhohighly expressed fusion protein Stx2 B-GST induced byIPTG. It was identified by SDS-PAGE, and was purified by GST-Sepharose Resin. Theantisera were prepared respectively by immunizing rabbits (New Zealand White) withpurified fusion pro
12、tein Stx2B and autoclaved E.coli O157 ATCC 43889.The antisera to Stx2B and E.coli O157 were purified by differential(NH4)2SO4-precipitated,caprylicacid-(NH4)2SO4precipitation,andproteinGchromatography respectively. Among the three methods, it showed that the purified IgGagainst Stx2B by the protein
13、G chromatography have a better effects and easier to preparea stable complex of rabbit polyclonal antibody against Stx2B conjugated colloidal goldbeads. Otherwise, the comparison showed caprylic acid-(NH4)2SO4 precipitation to purifyantisera to E.coli O157 was better than the other two methods.Accor
14、ding specific lateral- flow and colloidal gold tracing function associated withimmunology principle, we established and optimized the sandwich GICA to detect E.coliO157 and Stx2. We optimized reaction system for labelling-antibody in which the optimalamount of the purified anti-Stx2B IgG conjugated with colloidal gold beads was 60 g/mL,the purified antibody for E.coli O157 was 57g/mL, the optimal pH was 8.2, the size ofcolloidal gold partical was 20nm, the optim
