【摘要】：口腔和咽喉疾病是人類的常見(jiàn)疾病,主要是由于細(xì)菌感染所引起的,經(jīng)過(guò)研究測(cè)定,主要的感染菌為鏈球菌、葡萄球菌和肺炎雙球菌等。雖然口腔和咽喉疾病不會(huì)馬上引起人類死亡,但由于其病種多樣化,容易復(fù)發(fā),難以根治,且由其所引發(fā)的許多并發(fā)癥給人類的生活和工作帶來(lái)很多不便和麻煩。因此,關(guān)于口腔及咽喉疾病的治療一直受到人們的關(guān)注。 目前,人類抗細(xì)菌及病毒主要依賴抗生素,但是由于抗生素治療會(huì)產(chǎn)生很多不良后果,包括身體機(jī)能協(xié)調(diào)機(jī)制的紊亂化副作用,細(xì)菌、病毒的耐藥性幾率增加等。近年來(lái),人們開(kāi)始從免疫學(xué)的角度進(jìn)行研究,發(fā)現(xiàn)用IgY技術(shù)可以產(chǎn)生針對(duì)耐藥菌的抗體,可治療由細(xì)菌或病毒感染引起的疾病,具有安全無(wú)殘留、不產(chǎn)生抗藥性、價(jià)廉等優(yōu)點(diǎn),是一種具有廣闊應(yīng)用前景的抗生素替代品。 本研究制備了抗肺炎鏈球菌、MG溶血性鏈球菌特異性卵黃抗體,然后通過(guò)SDS-PAGE凝膠電泳、考馬斯亮藍(lán)染色法、抑菌試驗(yàn)、酶聯(lián)免疫吸附試驗(yàn)、交叉反應(yīng)試驗(yàn)對(duì)其生物活性進(jìn)行鑒定,并對(duì)IgY的熱穩(wěn)定性、酸堿穩(wěn)定性以及對(duì)消化酶的穩(wěn)定性進(jìn)行了研究。 結(jié)果表明,乙酸-乙酸鈉緩沖液、PEG 6000和硫酸銨溶液三步法提純得到的IgY純度可達(dá)到95%左右；間接酶聯(lián)免疫吸附試驗(yàn)(ELISA)表明,母雞首次免疫10d后,卵黃中即有抗體產(chǎn)生,初次免疫20 d后效價(jià)逐步上升,高效價(jià)出現(xiàn)在初次免疫后40 d-80 d,抗肺炎鏈球菌特異性卵黃抗體的效價(jià)最高可以達(dá)到1：5120,抗MG溶血性鏈球菌特異性卵黃抗體的效價(jià)最高可以達(dá)到1：2560；體外抑菌實(shí)驗(yàn)發(fā)現(xiàn),特異性IgY對(duì)肺炎鏈球菌、MG溶血性鏈球菌能夠顯著抑制,抑菌率可達(dá)到90%以上;交叉反應(yīng)試驗(yàn)說(shuō)明制備的特異性IgY對(duì)其抗原菌能夠特異性結(jié)合。 綜上,本研究成功制備出高含量、高純度、特異性強(qiáng)的抗肺炎鏈球菌、抗MG溶血性鏈球菌特異性IgY,體外抑菌實(shí)驗(yàn)研究表明其能夠有效與細(xì)菌結(jié)合,并對(duì)其抗原菌具有很好的抑制作用,為特異性IgY在咽喉炎的防治應(yīng)用中奠定了基礎(chǔ)。
[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.
【學(xué)位授予單位】：廣東工業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R392

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