【作者】 冯瑜菲；

【导师】 师东方；

【作者基本信息】 东北农业大学 ， 预防兽医学， 2011， 硕士

【摘要】 猪水肿病(Edema disease of swine,ED)是由某些溶血性大肠杆菌引起断奶仔猪的一种毒血症。临床特征为头部和胃壁等处水肿、共济失调、惊厥和麻痹等。该病发病急、死亡快,往往来不及治疗,死亡率很高。猪水肿病的发生与大肠杆菌的F18ab菌毛与志贺毒素Ⅱ型变异体(Stx2e)密切相关。目前,对该病的防治主要采用大肠杆菌多价苗免疫和抗生素治疗,但防治效果均不理想。卵黄抗体作为一类可替代抗生素的高效生物防治制剂在防治猪水肿病中显示出明显的效果,为该病的防治开辟了新的途径。本研究提取了F18ab菌毛蛋白与Stx2e,并利用原核表达技术,分别获得了F18ab菌毛与Stx2e的具有中和抗原表位的Fed F重组蛋白和Stx2eB重组蛋白。分别以上述4种蛋白作为免疫原对6月龄海兰蛋鸡进行免疫。共进行3次免疫,每次间隔两周。其中,Stx2e免疫组在一免时分为0.20 mg /只和0.40mg/只两个免疫剂量组,二免时,将一免的每个免疫剂量组再分为两小组,两个小组的免疫剂量分别为该组一免时的免疫剂量和一免时免疫剂量的2倍,三免时,所有免疫组的免疫剂量均为二免时最高免疫剂量组的剂量;Stx2eB重组蛋白免疫组在一免时分为0.50 mg /只和1.00mg/只两个免疫剂量组,二免和三免的免疫方式同Stx2e免疫组;F18ab菌毛蛋白免疫组在一免时分为0.38 mg /只和0.76mg/只两个免疫剂量组,二免和三免的免疫方式同Stx2e免疫组;Fed F重组蛋白免疫组三次免疫的剂量分别为1.00mg/只、2.00mg/只、2.00mg/只。二免后一周收蛋并用氯仿抽提法提取卵黄抗体,用间接ELISA法检测抗体效价。其中,以Stx2e、Stx2eB重组蛋白、F18ab菌毛蛋白、Fed F重组蛋白作为免疫原的免疫组ELISA效价最高值分别为1:5120、1:5120、1:20480、1:1280,且高水平效价分别可维持6周、2周、4周、3周。安全性实验结果显示,4种卵黄抗体均达到《中国兽药典》对兽医生物制品的标准。吸附和吸附抑制试验结果显示,抗F18ab菌毛蛋白卵黄抗体和抗FedF重组蛋白卵黄抗体分别与F18ab+大肠杆菌按1:1的体积比混合作用30min后,均能够抑制F18ab+大肠杆菌对仔猪小肠上皮细胞的吸附,而未用卵黄抗体处理的F18ab+大肠杆菌可紧密的吸附于仔猪小肠上皮细胞表面。体外中和试验结果显示,抗Stx2e卵黄抗体和抗Stx2eB重组蛋白卵黄抗体均可以抑制Stx2e对Vero细胞的致病变作用,接种这2种卵黄抗体的Vero细胞和对照组相比仍有大量正常生长的细胞,仅出现少量的死亡细胞。体内中和试验结果显示,抗Stx2e卵黄抗体在原液、1:2、1:4三个稀释浓度下,对昆明鼠的保护率分别为100%、60%、60%,抗Stx2eB重组蛋白卵黄抗体在原液、1:2、1:4三个稀释浓度下,对昆明鼠的保护率分别为100%、60%、40%,而生理盐水对照组和仅腹腔注射卵黄抗体的对照组昆明鼠生长正常,仅注射Stx2e对照组昆明鼠在48h内死亡率达到80%。本研究还建立了环介导等温扩增法(LAMP)用来检测Stx2e基因,根据GenBank中12条Stx2e序列和9条Stx2序列,针对Stx2e基因的保守区设计一套引物。以煮沸法处理的Stx2e+大肠杆菌过夜培养物为模板进行LAMP反应,所得扩增产物溶液变浑浊而对照管未发生变化。LAMP扩增产物电泳后呈特征性梯状条带,而阴性对照无扩增条带,在LAMP体系中加入1μl SYBR GreenⅠ染料后,肉眼观察时阳性管呈翠绿色,而阴性管则为淡橙色,紫外灯下,阳性管发出绿色荧光,而阴性管则无颜色变化。经反应条件优化后的LAMP方法在63℃反应1h后可得最佳反应效果。特异性试验结果显示,该LAMP方法仅对Stx2e+大肠杆菌产生特征性梯状扩增条带,而对LT+、ST+、Stx2+大肠杆菌检测结果均为阴性。敏感性试验结果显示,该方法对Stx2e+大肠杆菌的最低检出量为38CFU/管,是PCR方法敏感性的100倍。LAMP方法和PCR方法对模拟样品检测结果的符合率为100%。证明本法具有快速、特异和敏感的特点。本研究结果表明,针对致猪水肿病大肠杆菌的Stx2e与F18ab菌毛可以制备出高效价的卵黄抗体,为该病的特异性防治提供了物质基础,并为下一步的临床应用提供了实验基础。所建立的针对Stx2e基因的LAMP快速检测方法具有快速、灵敏、特异、结果判定简便的特点,有利于猪水肿病的诊断和Stx2e+大肠杆菌的快速检测。更多还原

【Abstract】 Edema disease of swine (ED) is mainly caused by Hemolytic Escherichia coli which lead to edema of head, stomach submucosa, ataxia, spasm and tetraplegia. ED is an acute disease. Because the onset of disease is often sudden and the course rapid, treatment is often ineffective, and mortality rate is very high. Following the researches of these years, people become grasp the pathopoiesis mechanism of edema disease. F18ab fimbriae and Stx2e toxin were two pathogenic factors of edema disease. At present, Escherichia coli multivalent vaccine and antibiotics were used to prevent and treat this disease, but the expected effects were not well. Egg yolk antibody as a kind of effective alternative to antibiotics in biological control showed the obvious effect and also opened a new way in the prevention against to the edema disease of swine.In this study, F18ab fimbriae and Stx2e toxin were extracted, and the recombinant protein FedF and Stx2eB, which had neutralizing epitope were obtained respectively by prokaryotic expression technology. Then the four different proteins were used as immunogen to immunize the six months old Highland laying hens for three times. Each time interval was two weeks. Chickens were divided into four groups. The chicken of Stx2e toxin group were divided into two groups and each group were immunized 0.20mg per chicken and 0.40mg per chicken respectively in the first immunization. Two weeks later, the second immunity was performed. Then the two groups of first immunization were divided into two subgroups respectively, the dose of subgroups were the same as the first immunization and twice as the first immunization respectively. In the third immunization, each chicken was immunized as much as the highest dose of the second immunization. The chicken of Stx2eB recombinant protein group were divided into two groups and each group were immunized 0.50mg per chicken and 1.00mg per chicken respectively in the first immunization. The second and the third immunization were immunized with the same method as Stx2e toxin group. The chicken of F18ab fimbriae protein group were divided into two groups and each group were immunized 0.38mg per chicken and 0.76mg per chicken respectively in the first immunization. The second and the third immunization were immunized with the same method as Stx2e toxin group. The dose of FedF recombinant protein group of three immunity times was 1.00mg per chicken, 2.00mg per chicken and 2.00mg per chicken respectively. One week after the second immunization, the eggs were collected and the yolk antibodies were extracted, then the titers of the yolk antibodies were detected by indirect ELISA. The highest titer of the Stx2e toxin group was 1:5120. The highest titer of the Stx2eB recombinant protein group was 1:5120. The highest titer of the F18ab fimbriae protein group was 1:20480. The highest titer of FedF recombinant protein group was 1:1280.And the high level of titers could maintain for six weeks, two weeks, four weeks and three weeks respectively. The results of safety experiment showed that egg yolk antibodies reached the standard of biological product from Chinese Veterinary Pharmacopoeia. In vitro tests showed that E. coli (F18ab+) adhesion to small intestine epithelial cells was inhibited with appropriate anti-F18ab fimbriae antibody or anti-FedF recombinant protein antibody preparations by preincubating the mixture of 1 ml of bacteria and 1 ml of antibody solution for 30 min, however, E. coli (F18ab+) without pretreatment with antibody adhered to the epithelia cells surfaces. The neutralization test in vitro showed that both anti-Stx2e toxin egg yolk antibody and anti-Stx2eB recombinant protein egg yolk antibody could inhibit the pathogenic affection to Vero cell. Comparing with control group the groups with appropriate anti-Stx2e toxin or anti-Stx2eB egg yolk antibody had little dead cell instead of most normal growth cell. The neutralization test in vivo showed that anti-Stx2e toxin egg yolk antibody stock solution, 1:2 dilution and 1:4 dilution had protection to Kunming mice and the rate of protection was 100%, 60% and 60% respectively. So did the anti-Stx2eB recombinant protein egg yolk antibody to Kunming mice and the rate of protection was 100%, 60% and 40% respectively. While the mouse which were injected physiological saline or egg yolk antibody only were normal growth and the mouse which were injected Stx2e toxin only were dead in 48h. The mortality was 80%.This study also established a loop-mediated isothermal amplification (LAMP) method for the detection of Shiga-like toxin II variant producing Escherichia coli. According to 12 Stx2e sequences and 9 Stx2 sequences available in GenBank, a set of four primers were designed based on the Stx2e conservative sequence. DNA was extracted from an overnight culture of the E. coli (Stx2e+) by boiling method to do LAMP assay. Product of positive reaction with LAMP became turbid and the negative control was no change. Product of positive reaction with LAMP showed ladder-like pattern on gel electrophoresis and the negative control had no ladder-like pattern. After addition of SYBR Green I, the products can be visualized directly by the naked eye or under UV light, which showed green while the negative control showed light orange. The optimal reaction temperature and time of the LAMP were investigated. LAMP assays, which were under isothermal condition of 63℃for 1h had the best results. The specificity test showed that ladder-like products were produced with those Stx2e positive samples by LAMP, while no product was generated with other controls(LT+、ST+、Stx2+). The sensitivity assay of LAMP had a detection limit equivalent to 38cfu/tube, was 100 times sensitive than PCR method (3.8×103cfu/tube). The agreement rate between LAMP and PCR was 100% in detecting simulation samples. Thus the LAMP assay was a rapid, specificity and sensitivity method.Results of this study indicated that the preparations of egg yolk antibodies could be produced aimed at Stx2e toxin and F18ab fimbriae. And these offered material basis for preventing edema and experiment basis for applying in clinical setting. The LAMP assay was rapid, sensitive, specific and judging easily so that it had the potential to be developed into a rapidly diagnosis tool for edema disease of swine and detection of Escherichia coli producing Stx2e.更多还原