抗肠道病毒71型特异性卵黄抗体的制备及鉴定

摘要

0bjsctive iPreparhg specificity chicken egg yolk antibody (IgY ) against enteiovius 71 (EV 71) and detecting its biobgical activities,this is significantfor prevention and treatnentof EV 71 infection .M ethods:Fourhensweie inmurmed Ihree tineswiih tie purified him an EV71 virus as an antigen .The inmunogbbulh Y (IgY ) antibody was extracted and purified from tie egg yolk by water combine pofyeihylene gfycol (water-peg) m eihod .The yield and molecularm ass of IgY were analysed by Bradford m eihod and SDS-PAGE electrophore- sis.ELBA and A gar double in munodiffusbn were used for detect 1he titer of IgY ,while tie inmunoreactivily of antHiV71 IgY was tested using W estem bbt.Results :The yield and purily of IgY were 7 .76 m g/m land 94.86% respective^ .Results of ELBA hdicated 1hat1he specific an- trEV71 IgY antibody appealed in tie egg yolk 10 days afterprinary inmuniation and reached 1he highest titer 1 = 20 480 after 40 days,while 1he titer of A gar double in m unodiffusion was 1 = 16 .W estem bbt analysis revealed lhattie purified IgY antibody owned good inmunoreactivily and a specifically combination capacily of EV71 virus.Conclusbn :Egg yolk is a large scale source of antidodys specifically against EV 71 virus .The antrEV 71 IgY extracted and purified by W aterPEG m etiod possess good production ,purtly ,and bbbgical activities.%目的:制备特异性抗肠道病毒71型(Enterovirus 71,EV71)的卵黄抗体并检测其生物学性能,这对EV71感染手足口病的预防和治疗具有重大的意义.方法:用纯化并灭活后EV71作为抗原免疫健康产蛋鸡.采用本实验室水提综合聚乙二醇法提取纯化鸡卵黄抗体;用Bradford法测定卵黄抗体提取液的蛋白含量;还原性SDS-PAGE凝胶电泳分析测定提取蛋白的纯度及相对分子质量;分别用ELISA、双向免疫琼脂扩散检测纯化特异性卵黄抗体抗EV71效价;Western blot分析特异性卵黄抗体的免疫反应性.结果:卵黄中卵黄抗体含量达7.76 mg/ml,卵黄抗体的纯度为94.86%.初免10天后蛋黄中可检测到特异性抗EV71卵黄抗体,初免40天后ELISA检测抗体效价高达1∶20 480,双向琼脂扩散检测效价达1∶16.提取的卵黄抗体具有良好的免疫反应性,能与EV71特异性结合.结论:水提综合聚乙二醇法提取纯化得到的抗EV71卵黄抗体产量和纯度高、效价好且具有良好的免疫反应性.