【作者】 徐敬宜；

【导师】 徐永平；

【作者基本信息】 大连理工大学 ， 生物化工， 2006， 硕士

【摘要】 口服特异性卵黄免疫球蛋白（Egg Yolk Immunoglobulins,IgY）可治疗由细菌或病毒引起的肠道疾病,具有安全无残留、不产生抗药性、成本低和服用简便等优点,是一种具有广阔应用前景的抗生素替代品。但IgY容易在高强度的胃酸环境中被胃蛋白酶降解,失去抗体活性。因此,如何避免IgY在胃中失活,在小肠中稳定地发挥作用,是一个亟待解决的问题。 本研究采用高压静电成囊技术,选取天然材料海藻酸钠（Alginate,ALG）和壳聚糖（Chitosan,CTS）作为囊材,制备口服型IgY-ALG-CTS微囊,使IgY在胃中受到保护而免受降解,并在肠道中定点释放。本研究具体考察了各种物理参数对空白微囊形貌及粒径的影响;并在此基础上研究了投药量、ALG浓度、CTS浓度、CaCl₂浓度及CTS溶液的pH对微囊的包封率、IgY的活性、以及微囊在模拟胃肠液中释放行为等性能指标的影响。 对空白微囊的研究结果表明,外加电场强度是影响微囊粒径的主要因素,泵的推进速度和ALG、CTS、CaCl₂溶液的浓度主要影响微囊的机械强度,对微囊粒径的影响不大。优化条件为:外加电压6kv,针头液面距40mm,推进速度60ml/h,ALG浓度1.8%,CaCl₂浓度0.5%,CTS浓度0.4%。优化条件下制得的微囊球形圆整,冷冻干燥后平均粒径为152.28μm,且粒径分布集中。 对IgY微囊的研究结果表明,微囊制备的优化条件为:投药量10mg/ml,CaCl₂浓度0.1%,CTS浓度0.4%,ALG浓度2%,CTS溶液的pH为4。在此优化条件下制备的IgY微囊包封率高于90%,IgY在模拟胃液中累积释放率小于10%,而在模拟肠液中累积释放率大于90%。另外,ELISA和SDS-PAGE结果显示,IgY微囊在通过模拟胃液后进入模拟肠液,释放出完整的IgY,而且抗体的活性保持在80%以上。达到了在胃中保护IgY免受胃蛋白酶降解,使之以较高活性在小肠释放的目的。 以ALG和CTS为囊材制备的IgY微囊,作为一种新型的口服动物保健产品,将具有良好的产业化前景。本研究也为其他口服生物活性物质的控释制剂开发奠定了基础。更多还原

【Abstract】 Chicken egg-yolk immunoglobulin （IgY） provides an inexpensive and effective source of antibodies for the passive immunization of animals suffering from intestinal diseases. It is a promising alternative for the treatment and prevention of enteric infections, and has shown to be effective against a number of gastrointestinal pathogens, including Escherichia coli, Salmonella and rotavirus. However, its application is limited by its sensitivity to gastrointestinal conditions.Here, the IgY-containing microcapsules are made with nature materials-Alginate （ALG） and Chitosan （CTS）. High-voltage electrostatic system is used to prepare ALG-CTS microcapsules for IgY via electrostatics interaction.For the blank microcapsules, optimum operating conditions are established for preparation of homogenous, spherical, and smooth alginate-chitosan microcapsules. The optimum results can be obtained under the following conditions: voltage 6 kv, distance from needle end to fluid 40 mm, pump rate 60 ml/h, CaCl₂ concentration 0.5%, ALG concentration 1.8%, CTS concentration 0.4%. The mean diameter of the microcapsules prepared under the optimum conditions is 152.28 μm.For the IgY microcapsules, optimum results are obtained under the following conditions: IgY loading rate 10 mg/ml, CaCl₂ concentration 0.1%, ALG concentration 2%, CTS concentration 0.4%, CTS pH 4. The entrapment efficiency, IgY activity retained and in vitro release of the IgY prepared under the optimum conditions in SGF （Simulated Gastric Fluid） and SIF （Simulated Intestinal Fluid） are examined. IgY entrapment efficiency achieves 90% in the optimum condition. The IgY retains 80% activity after 2 h exposure to SGF and then 6 h exposure to SIF. Less than 10% IgY is released upon the microcapsules exposure to SGF for 2 hours, and more than 90% IgY is released upon the microcapsules exposure to SIF for 6 h. Electrophoretic studies of IgY microcapsules passed gasintestinal tract show the intact protein band.Consequently, it can be said that the system can be offered for the temporary protection of IgY against acidic and enzymatic degradation during gastric passage, enabling its use for oral passive immunotherapy.更多还原