离子交换色谱法分离纯化鸡卵黄免疫球蛋白

建立了高效、经济、大规模获得鸡卵黄免疫球蛋白(IgY)的生产方法。在对传统的水稀释法改良的基础上,结合聚乙二醇沉淀与离子交换色谱进行IgY的分离纯化。结果显示,用8倍无菌水稀释蛋黄液,用0.1 mol/L HCl调节pH为5.2,在4 ℃下静置8 h,于5000×g力离心可得上清粗IgY液,经测定回收率可达93.47%。然后用6%聚乙二醇沉淀后经DEAE-Toyopearl 650 M离子交换纯化,最佳的纯化条件: 0.05 mol/L磷酸盐缓冲液(PBS, pH 7)平衡上样,0.075 mol/L PBS(pH 7)洗脱。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分析结果显示所得的IgY的纯度为95.02%,活性保持率高达73.77%。本研究弥补了传统分离方法不能同时达到高纯度和高回收率的缺点,且可用于大规模生产。

Preparation of immunoglobulin from chicken egg yolk by anion-exchange chromatography

An economical, effective and large-scale method for the preparation of immunoglobulin of chicken egg yolk (IgY) was developed. The method was based on an improved water dilution combining with polyethylene glycol 6000 (PEG6000) precipitation and anion-exchange chromatography. The egg yolk was diluted with 8 volumes of aseptic water, the pH of which was adjusted to 5.2 with 0.1 mol/L HCl, then the mixture was centrifuged at 5 000 x g after standing for 8 h at 4 degrees C. The crude IgY was obtained from the supernatant layer with the yield of 93.47%. The purification of IgY was achieved by 6% PEG6000 precipitation and DEAE-Toyo-pearl 650 M anion-exchange chromatographic packing. The optimum purified conditions by ion-exchange chromatography were as follows: equilibration with 0.05 mol/L phosphate buffer (pH 7.0) and elution with 0. 075 mol/L phosphate buffer (pH 7.0). The purity of the IgY fraction was consistently greater than 95% with high activity (73.77%) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). This method is superior to the traditional ion-exchange chromatography that can't simultaneously obtain high purity and high recovery. Moreover, this method is suitable for a large-scale IgY preparation from chicken egg yolk.