抗咽喉炎链球菌鸡卵黄抗体的制备及检测

发布时间：2018-11-04 08:38

【摘要】：口腔和咽喉疾病是人类的常见疾病,主要是由于细菌感染所引起的,经过研究测定,主要的感染菌为链球菌、葡萄球菌和肺炎双球菌等。虽然口腔和咽喉疾病不会马上引起人类死亡,但由于其病种多样化,容易复发,难以根治,且由其所引发的许多并发症给人类的生活和工作带来很多不便和麻烦。因此,关于口腔及咽喉疾病的治疗一直受到人们的关注。目前,人类抗细菌及病毒主要依赖抗生素,但是由于抗生素治疗会产生很多不良后果,包括身体机能协调机制的紊乱化副作用,细菌、病毒的耐药性几率增加等。近年来,人们开始从免疫学的角度进行研究,发现用IgY技术可以产生针对耐药菌的抗体,可治疗由细菌或病毒感染引起的疾病,具有安全无残留、不产生抗药性、价廉等优点,是一种具有广阔应用前景的抗生素替代品。本研究制备了抗肺炎链球菌、MG溶血性链球菌特异性卵黄抗体,然后通过SDS-PAGE凝胶电泳、考马斯亮蓝染色法、抑菌试验、酶联免疫吸附试验、交叉反应试验对其生物活性进行鉴定,并对IgY的热稳定性、酸碱稳定性以及对消化酶的稳定性进行了研究。结果表明,乙酸-乙酸钠缓冲液、PEG 6000和硫酸铵溶液三步法提纯得到的IgY纯度可达到95%左右；间接酶联免疫吸附试验(ELISA)表明,母鸡首次免疫10d后,卵黄中即有抗体产生,初次免疫20 d后效价逐步上升,高效价出现在初次免疫后40 d-80 d,抗肺炎链球菌特异性卵黄抗体的效价最高可以达到1：5120,抗MG溶血性链球菌特异性卵黄抗体的效价最高可以达到1：2560；体外抑菌实验发现,特异性IgY对肺炎链球菌、MG溶血性链球菌能够显著抑制,抑菌率可达到90%以上;交叉反应试验说明制备的特异性IgY对其抗原菌能够特异性结合。综上,本研究成功制备出高含量、高纯度、特异性强的抗肺炎链球菌、抗MG溶血性链球菌特异性IgY,体外抑菌实验研究表明其能够有效与细菌结合,并对其抗原菌具有很好的抑制作用,为特异性IgY在咽喉炎的防治应用中奠定了基础。
[Abstract]:Oral and throat diseases are common diseases in human, mainly caused by bacterial infection. The main infection bacteria are Streptococcus, Staphylococcus and pneumococcus. Although oral and throat diseases do not immediately cause human death, but because of its variety of diseases, easy to relapse, difficult to cure, and many complications caused by the human life and work brought a lot of inconvenience and trouble. Therefore, the treatment of oral and throat diseases has been paid attention to. At present, human anti-bacteria and virus mainly rely on antibiotics, but because of antibiotic treatment will have many adverse consequences, including the disorder side effects of the coordination mechanism of body function, bacteria, virus drug resistance probability increased and so on. In recent years, people began to study from the perspective of immunology, and found that IgY technology can produce antibodies against drug-resistant bacteria, can treat diseases caused by bacterial or viral infection, and has the advantages of safety and no residue, no drug resistance, low price, and so on. It is a promising substitute for antibiotics. In this study, specific yolk antibodies against Streptococcus pneumoniae and MG hemolytic streptococcus were prepared, followed by SDS-PAGE gel electrophoresis, Coomassie brilliant blue staining, bacteriostatic test and enzyme-linked immunosorbent assay (Elisa). The biological activity of IgY was identified by cross reaction test. The thermal stability, acid-base stability and digestive enzyme stability of IgY were studied. The results showed that the purity of IgY obtained by three-step purification of acetic acid-sodium acetate buffer solution, PEG 6000 and ammonium sulfate solution was about 95%. Indirect enzyme-linked immunosorbent assay (ELISA) showed that antibody was produced in the egg yolk 10 days after the first immunization, and the titer increased gradually after the first immunization for 20 days, and the high titer appeared at 40 d to 80 d after the first immunization. The titer of specific yolk antibody against Streptococcus pneumoniae was 1: 5120, and the titer of specific egg yolk antibody against MG hemolytic streptococcus could reach 1: 2560. In vitro bacteriostatic test showed that specific IgY could inhibit Streptococcus pneumoniae and MG hemolytic streptococcus significantly, and the inhibition rate could reach more than 90%, and the cross reaction test showed that the specific IgY could bind specifically to the antigenic bacteria of Streptococcus pneumoniae. In conclusion, we successfully prepared high content, high purity and strong specificity of anti Streptococcus pneumoniae and anti MG hemolytic streptococcus IgY, in vitro. It also has a good inhibitory effect on the antigenic bacteria, which lays a foundation for the application of specific IgY in the prevention and treatment of pharyngitis.
【学位授予单位】：广东工业大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392

【相似文献】