【作者】 李晓宇；

【导师】 徐永平；

【作者基本信息】 大连理工大学 ， 生物化工， 2008， 博士

【摘要】 口服特异性卵黄免疫球蛋白（Egg yolk immunoglobulin,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、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在高强度的胃酸环境中易被胃蛋白酶降解,失去抗体活性。因此,如何避免IgY在胃中失活,而在小肠中稳定地发挥作用,是一个亟待解决的问题。针对这一问题,本论文采用微囊化技术包埋IgY,制备口服型IgY-壳聚糖-海藻酸钠微囊,从体外、体内两方面评价壳聚糖-海藻酸钠微囊作为IgY口服载体的可行性。本研究以引起仔猪腹泻的最主要致病菌——产肠毒素大肠杆菌K88为抗原,免疫蛋鸡后获得特异性IgY。通过对获得高效价特异性IgY的最佳抗原浓度研究发现,K88菌毛蛋白对蛋鸡的免疫原性优于全菌;纯化的菌毛蛋白对蛋鸡的免疫原性优于粗菌毛蛋白,其诱导的抗体效价最高可达1:480000;确定菌毛蛋白的最佳浓度为2 mg/ml,全菌的最佳浓度为10¹⁰cfu/ml。通过对IgY的分离纯化条件研究发现,盐析法提取IgY的最佳条件是将粗提的IgY经50%饱和度的硫酸铵盐析一次,再经14%（w/v）硫酸钠盐析一次,获得的IgY回收率可达46%,纯度可达77%;经超滤（100 kDa）后,最终获得的IgY冻干粉纯度为82%。对IgY稳定性的研究结果表明,温度不超过70℃时,IgY具有良好的热稳定性;pH＜3时,IgY活性大部分丧失;IgY对胰蛋白酶有较好的抵抗力,但低pH条件下对胃蛋白酶十分敏感;冷冻干燥处理对IgY活性有不利的影响;-20℃和4℃环境中,IgY冻干粉具有良好的贮藏稳定性。为保护IgY的口服活性,选用天然材料海藻酸钠和壳聚糖作为壁材,利用气体吹喷制囊法,制备IgY-壳聚糖-海藻酸钠微囊。考察了成囊装置主要的物理条件对空白壳聚糖-海藻酸钠微囊粒径及形态的影响,确定制备微囊的最佳物理条件为:空气压力0.3m³/h,,液面距8 cm,恒流泵速度4 ml/min,搅拌子转速200 r/min。此条件下制备的新鲜微囊球形圆整,平均粒径约1 mm,且粒径分布集中。在此基础上,探讨了主要反应条件对微囊的载药量、包封率、保护IgY在模拟胃液中的稳定性、以及微囊在体外的释放行为的影响。确定制备微囊的优化条件为:成囊溶液pH 3.5,壳聚糖浓度0.2%（w/v）,CaCl₂浓度0.5%（w/v）,海藻酸钠浓度2%（w/v）,药载比25%,在此优化条件下制备的IgY微囊载药量高于20%,包封率高于75%,IgY在模拟胃液中2 h累积释放率小于10%,而在模拟肠液中4 h累积释放率大于80%。另外,ELISA和SDS-PAGE结果显示,IgY微囊在模拟胃液中可抵抗胃蛋白酶的降解,而且抗体的活性保持在70%以上。将IgY-壳聚糖-海藻酸钠微囊用于断奶仔猪的攻毒治疗试验,结果表明,腹泻断奶仔猪服用IgY微囊后,病情很快得到控制,1 d后所有病猪得到治愈,而且体重增加最快;而未包埋的IgY冻干粉治疗效果低于微囊化IgY,3 d后所有病猪得到治愈。而对照组仔猪口服生理盐水,第1 d有腹泻症状的仔猪3 d后仍未见好转,体重下降。由此可见,IgY经壳聚糖-海藻酸钠微囊包埋后,可以很好的保护其在胃酸环境中的抗体活性,并且可于小肠中定点释放,能有效治愈断奶仔猪腹泻。综上,本研究制备的IgY微囊,可保证IgY以较高的活性到达小肠,并在肠道中定点释放,稳定的发挥作用。其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 gastric conditions.Here, IgY-loaded microcapsules were prepared using nature polysaccharides such as alginate and chitosan. The objective of this study was to evaluate chitosan-alginate microcapsules as a method of oral delivery of IgY antibodies in vitro and in vivo.Enterotoxigenic Escherichia coli K88 （ETEC K88） was used as an antigen to immunize laying hens, and then produced specific IgY. Immunogenicity of laying hens was investigated following immunization with different antigen preparations, including crude fimbriae, purified fimbriae and formalin-killed whole cells. The results showed that the antibody titer was much greater when the fimbriae were used as an antigen （480000） compared with that of antibodies with the formalin-killed bacteria （320000）. Moreover the purified fimbriae dramatically increased the titer. The optimal concentration of purified fimbriae and formalin-killed bacteria was found to be 2 mg/ml and 10¹⁰ cfu/ml respectively, which resulted in the greatest and most stable antibody titer.The optimal concentrations of salt were investigated for purification of IgY by a two-step procedure using salt precipitation. The results showed that precipitation with 50% saturation ammonium sulfate followed by precipitating with 14% sodium sulfate gave optimum IgY recovery （46%） and purity （77%）. Upon ultrafiltration, the purity of final IgY powder was 82%.IgY was fairly heat-stable and most of the antibody activity remained after heating up to 70℃for 15 min. IgY was stable at pH above 3, but was very susceptible to pepsin in low pH, while appreciable activity was retained by IgY after trypsin digestion. Freeze-drying could affect activity of IgY. Moreover, IgY was stable stored at -20℃or 4℃for 12 months.Air-driven droplet generator was used to prepare IgY-loaded chitosan-alginate microcapsules. The effects of physical-chemical factors on the properties of IgY-loaded microcapsules were investigated. The properties investigated included the loading capacity for IgY （expressed as the IgY loading percentage, %, w/w of microcapsules）, encapsulation efficiency （EE%）, gastric stability and release characteristics of these microcapsules. Optimum physical factors were established for preparation of homogenous, spherical and smooth microcapsules. For the IgY-loaded microcapsules, optimum results were obtained under the following conditions: pH of the encapsulation medium 3.5, chitosan concentration 0.2% （w/v）, alginate concentration 2% （w/v）, CaCl₂ concentration 0.5% （w/v）, IgY loading rate 25% （w/w）. Under the optimum conditions, IgY loading% and EE% was above 20% and 75%, respectively. The stability of IgY in simulated gastric fluid （SGF, pH 1.2） was greatly improved by encapsulation in chitosan-alginate microcapsules, and retained 70% activity after 2 h exposure to SGF. Moreover, microencapsulated IgY was significantly resistant to pepsin hydrolysis. Less than 10% IgY was released upon the microcapsules exposure to SGF for 2 h, and more than 80% IgY was released upon the microcapsules exposure to simulated intestinal fluid （SIF, pH 6.8） for 4h.The protective effects of microencapsulated IgY obtained from hens immunized with ETEC K88 fimbrial antigen were evaluated in 40-day-old piglets in which ETEC diarrhea was induced. The results demonstrated that the ETEC K88 induced diarrhea was cured 24 h after treated with microencapsulated IgY, and the body weight gain was the fastest during the period of the experiment while those treated with non-encapsulated IgY were cured within 72 h after infection. Whereas control piglets treated with normal saline had severe diarrhea and dehydration after infection. These results indicated that the stability of IgY under gastric conditions was greatly improved by encapsulation in chitosan-alginate microcapsules, and weaned piglets that received microencapsulated IgY were better protected against ETEC infection.Consequently, it can be said that chitosan-alginate microcapsules 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