【作者】 王通；

【导师】 徐永平；

【作者基本信息】 大连理工大学 ， 生物化工， 2020， 硕士

【摘要】 产肠毒素大肠杆菌(Enterotoxigenic E.coli,ETEC)是养殖业最常见能够导致仔猪腹泻的病原体之一。新生仔猪由于肠道较为脆弱,免疫机能发育不完全,极易受到各类病原体的侵袭。ETEC导致的仔猪大肠杆菌性腹泻伴有多种症状,其主要表现为仔猪黄白痢,受腹泻影响的仔猪通常还会出现虚弱脱水、精神萎靡、发育缓慢等问题。仔猪腹泻严重影响仔猪健康和生产性能,治疗不及时可导致仔猪生产性能下降,严重时可造成仔猪死亡。仔猪腹泻处理不及时可造成病原体在养殖环境内快速传播,影响生猪养殖规模,给养殖业造成巨大的经济损失。仔猪腹泻造成养殖成本增加,影响生猪供应,猪肉制品价格上涨,给消费者带来经济负担。本实验以产肠毒素大肠杆菌F4、F5、F6、F18、F41菌毛及STa-LTB-STb重组肠毒素为抗原,联合制备针对菌毛和肠毒素两大靶点的多价疫苗,制备特异性IgY,实现对两大毒力因子的双重阻断。免疫蛋鸡,收集卵黄液并利用水稀释-硫酸铵沉淀法结合超滤浓缩对卵黄抗体进行分离纯化。实验结果显示,抗体最终纯度达到84.8%,IgY对五种菌毛抗原的效价均超过1:10000,最高效价可达1:32000,对重组肠毒素的效价最高达1:8000,表明多价抗原具有良好的免疫原性,适于IgY的生产及制备。体外实验中,利用胶体金标记的兔抗鸡IgG特异性吸附卵黄抗体,在透射电镜下观察卵黄抗体同各抗原的结合效果,结果显示特异性卵黄抗体能同菌体表面紧密结合,同时被结合菌体表面发生结构改变。液体环境多价特异性卵黄抗体有效抑制K88、K99、987p、F18和F41等五种产肠毒素大肠杆菌增殖,其抑制效果具有浓度依赖性,抑菌效果随卵黄抗体浓度升高更加显著。以二甲苯和十六烷为有机相,借助碳氢吸附实验检验卵黄抗体对各株ETEC疏水性的影响,结果显示各菌体表面疏水性显著增强,与以往研究结果有所差别,为探明卵黄抗体的真实作用机制提供参考。动物实验中,以断奶SD大鼠为对象建立模型,以灌胃的方式进行攻毒和给药,分别检测特异性卵黄抗体和非特异卵黄抗体对ETEC的与预防和治疗效果。特异性IgY有效降低了大鼠的腹泻率,促进增重,改善生长发育情况,显著降低血液及组织中的TNF-α、IL-1β和IL-6水平(P<0.05),缓解由ETEC引起的炎症反应,保护肠道绒毛免受ETEC肠毒素的破坏,体现出良好的防治效果,实现了对菌毛和肠毒素的双重阻断,对大鼠肠道起到全方位的保护作用,为进一步探究卵黄抗体在动物体内的作用机制以及在养殖动物中的推广应用提供实验参考。更多还原

【Abstract】 Enterotoxigenic E.coli(ETEC)is one of the most common pathogens in the breeding industry that can cause diarrhea in piglets.Newborn piglets are vulnerable to various pathogens due to their weak intestines and incomplete development of immune function.ETEC-induced diarrhea in piglets caused by ETEC is accompanied by a variety of symptoms,mainly manifested as yellow and white diarrhea in piglets.Piglets affected by diarrhea often also suffer from dehydration,mental depression,and slow development.Piglet diarrhea seriously affects the health and performance of piglets.Inadequate treatment can lead to a decline in piglet performance and,in severe cases,piglet death.Incomplete treatment of piglet diarrhea can cause pathogens to spread quickly in the breeding environment,affect the scale of pig breeding,and cause huge economic losses to the breeding industry.Piglet diarrhea causes increased breeding costs,affects the supply of live pigs,and the price of pork products rises,bringing economic burden to consumers.In this experiment,enterotoxin-producing Escherichia coli F4,F5,F6,F18,F41 fimbriae and STa-LTB-STb recombinant enterotoxin were used as antigens to jointly prepare a multivalent vaccine against the two major targets of fimbria and enterotoxin.Sexual IgY,to achieve double blocking of two major virulence factors.Immunize laying hens,collect yolk fluid and separate and purify yolk antibodies by water dilution-ammonium sulfate precipitation method combined with ultrafiltration concentration.The experimental results show that the final purity of the antibody reaches 84.8%,the potency of IgY for the five fimbriae antigens exceeds 1:10000,the highest potency can reach 1:32000,and the potency for recombinant enterotoxin up to 1:8000,indicating that Multivalent antigen has good immunogenicity,suitable for IgY production and preparation.In the in vitro experiment,the rabbit anti-chicken IgG labeled with colloidal gold specifically adsorbed yolk antibody,and the binding effect of the yolk antibody to each antigen was observed under transmission electron microscope.The results showed that the specific yolk antibody can tightly bind to the cell surface and be bound at the same time Structural changes on the cell surface.The multivalent specific egg yolk antibody in the liquid environment can effectively inhibit the proliferation of five enterotoxigenic Escherichia coli such as K88,K99,987 p,F18 and F41.The inhibitory effect is concentration-dependent,and the bacteriostatic effect is more significant with the increase of the yolk antibody concentration.Using xylene and hexadecane as the organic phase,the effect of egg yolk antibody on the hydrophobicity of each strain of ETEC was tested by means of a hydrocarbon adsorption experiment.The results showed that the surface hydrophobicity of each cell was significantly enhanced,which was different from the previous research results.The actual mechanism of action of yolk antibodies provides a reference.In animal experiments,weaned SD rats were used as the object to establish a model,and the rats were challenged and administered by intragastric administration.The specific yolk antibody and non-specific yolk antibody were tested for the prevention and treatment of ETEC.Specific IgY effectively reduces the rate of diarrhea in rats,promotes weight gain,improves growth and development,significantly reduces the levels of TNF-α,IL-1β,and IL-6 in blood and tissues(P<0.05),relieving ETEC Inflammatory reaction,protect the intestinal villi from the destruction of ETEC enterotoxin,exemplify a good prevention and treatment effect,realize the double blocking of fimbria and enterotoxin,and play a comprehensive role in protecting the intestine of rats.To further explore the mechanism of yolk antibodies in animals and to promote the application in breeding animals to provide experimental reference.更多还原