特异性抗肠出血性大肠杆菌O157:H7内毒素鸡卵黄抗体的制备

目的:比较研究LPS和O-SP的免疫原性,制备出特异性抗内毒素(LPS)鸡卵黄免疫球蛋白(eggyolkimmunoglobulin,IgY),用于对E.coliO157:H7的免疫预防及检测。方法:分别用灭活E.coliO157:H7菌体、不同浓度内毒素(LPS)及O-特异性多糖链(O-SP)与不完全弗氏佐剂充分乳化后作抗原免疫临产蛋母鸡,以水稀释法从卵黄中提取抗体,阴离子交换色谱法实现对抗体的纯化,间接ELISA及双向免疫扩散检测抗体产生效价与活性。结果与结论:所制内毒素(LPS)和O-SP均有较好的免疫原性,刺激鸡体后可产生高效价抗体,灭活菌体、600μg/mlLPS、1200μg/mlLPS、2000μg/mlLPS、O-SP抗体产生效价最高可分别达1:32000、1:28000、1:32000、1:12000、1:40000。结合离子交换色谱,用0.185mol/L的离子强度可实现一步洗脱纯化抗体,制备出高纯度、高活性、特异性强的鸡卵黄免疫球蛋白IgY,为大肠杆菌O157:H7的感染防治提供了可靠的保证,在此实验基础之上可进一步探讨应用特异性IgY对大肠杆菌O157:H7的检测。

Preparation Study on Specific Egg Yolk Antibody against Endotoxin of Escherichia coli O157:H7

Objective: To produce specific chicken egg yolk immunoglobulin against lipopolysaccharide(LPS), used for preventing and detecting E.coli O157:H7 in comparison with the immunity of LPS and O-specific polysaccharide (O-SP). Methods: Hens, 25-week old, were immunized with E.coli O157:H7, LPS, O-SP respectively after being emulsified with incomplete Freuds’ adjuvant. By a simple method of water dilute of protein IgY to separate the antibody from egg yolk, purified by ion-exchange chromatograph, the IgY detected by SDS-PAGE and indirect-ELISA, and its immunologic competence was determined by double immunodiffusion. Results and Conclusion:The IgY titers of E.coli O157:H7 are obtained with 600μg/ml LPS, 1200μg/ml LPS and 2000μg/ml LPS, as O-SP respectively 1:32000, 1:28000, 1:32000, 1:12000 and 1:40000. It is purified by ion exchanging with 0.185mol/L THS washing. The immunity of lipopolysaccharide and O-SP is strong. The IgY anti-OSP produced by immunizing hens shows high titer, high purification and well specificity by chicken egg yolk immunoglobulin. The results provide scientific basis for preventing and infection curing by O157:H7 and further ways exploring of detecting O157:H7 with specific anti-LPS IgY produced from hens.