【作者】 谢献胜；

【导师】 王锋； 丁铲；

【作者基本信息】 南京农业大学 ， 养殖， 2005， 硕士

【摘要】 试验以幽门螺杆菌（Helicobacter pylori，Hp）为材料，采用不同培养基进行培养和保存，并制备成完全抗原对刚开产或开产不久的母鸡进行免疫，用凝集试验法测定其抗体效价，并对免疫Hp母鸡免疫应答规律和卵黄抗体的抑菌活性及理化特性做了初步探讨。为制备高含量的抗Up的免疫卵黄及相关生物的制剂提供技术依据，也为鸡蛋及免疫蛋的开发利用和深入研究积累经验并提供借鉴。 1 幽门螺杆菌的培养与保存 本试验采用不同培养基对幽门螺杆菌（Helicobacter pylori，Hp）进行培养和保存，结果表明：固体培养Hp，培养基为哥伦比亚琼脂基础加混合抗生素(10mg·L^-1盐酸万古霉素+10mg·L^-1两性霉素B+2500iu·L^-1多粘菌素B+5mg·L^-1甲氧苄胺嘧啶)，并分别添加6％脱纤维绵羊血、6％马血清，在5％O₂，10％CO₂，85％N₂的微需氧条件下，37℃培养3d，均可培养出Hp；严格无菌的条件下，不添加混合抗生素，对细菌的生长没有影响，且不会造成杂菌污染。液体培养Hp，布氏肉汤基础中加入混合抗生素和6％马血清，Hp增菌迅速，但如不及时更换新鲜的微需氧环境，极易形成球形体。菌株采取冷冻保存液法保存于-70℃～-80℃低温条件下6个月，可以成功复苏，且复苏率可达100％。 2 抗幽门螺杆菌卵黄抗体的制备及其体外抑菌试验 试验以幽门螺杆菌（helicobacter pylori，Hp）全菌灭活抗原对150日龄伊沙褐壳母鸡进行首免，165日龄二免，180日龄三免，于首免后即开始收集鸡蛋，采用凝集试验法测定卵黄中的抗体效价。结果表明：每间隔15天，采取两次加强免疫方法，可有效的引起免疫应答。母鸡在初免45d后，抗体效价可达到1:1024，同批样品在此水平可维持达60d以上（效价大于1:128），然后抗体效价呈缓慢下降趋势，经130d后，抗体效价下降至1:8。体外抑菌试验表明，高免卵黄具有明显的抑制HP生长的活性。 3 鸡抗幽门螺杆菌卵黄抗体的理化特性 试验对自制鸡抗幽门螺杆菌高免卵黄的理化特性进行了研究。结果发现：pH值、温度对其活性的影响较明显，胃蛋白酶则加强了这一作用。在pH值单独影响下，pH≤3为其敏感区，如果同时存在胃蛋白酶，则其敏感区为pH≤4，在巴氏消毒温度条件下其具有较好的热稳定性，这对于工业化生产卵黄抗体具有非常重要的意义。更多还原

【Abstract】 Different culture media were tested to culture and preserve Helicobacter Pylori （Hp）. The just mature chickens were procedurally immunized with complete Hp which cultured by the team, agglutination test were used to determine the egg yolk antibody level, and the pattern of immuno-responsion, bacteriostasis in Vitro, physical and chemical characteristic of immune yolk were searched. All these studies were of great significance to the industrial production of high immune yolk antibody against Hp and correlatively biologic medication.1 Effect of different methods on Hp culture and preservationThe strain was cultured on Columbia Agar Base （CAB） supplemented with mixed antibiotics containing 2500IU/l Polymyxin B Sulfate, 5mg/l Trimethoprim, 10mg/l Vancomycin HC1 and 10mg/l Amphotericin B and 6% defibrinated sheep blood or 6% horse serum respectively in a microaerophilic atmosphere of 5% O₂, 10% CO₂ and 85% N₂ at 37℃ for 72h. With the two methods, good effect was observed. In condition of sterilization with no supplementation with mixed antibiotics, the strain can’t be contaminated and grow well as well. When Brucella Broth was used an culture medium with combined antibiotics and horse serum in microaerophilic bag, Hp could grow rapidly but was easily to be spheroid without replacing the bag on time. And the liquid culture method was contaminated compared with solid method. The strain can be revived successively with cryopreservation at -70℃ to -80℃, 100 percents,2 Preparation and Bacteriostasis in Vitro of Egg Yolk Antibody Against Helicobacter PyloriPreparation and bacteriostasis in vitro of egg yolk antibody against Helicobacter pylori （Hp） have been studied. Chickens were procedurally immunized with complete bacterin at 150, 165 and 180 days old, successively. Agglutination test has shown that the egg yolk antibody against HP was produced from immunized hens, the antibody titer reached to 1:1024 at the 45th day after the first inoculability, the high level can last for 2 months, then go down gradually to 1:8 after about 130 days. Bacteriostasis in vitro shown that HP is hypersensitive to egg yolk antibody.更多还原