《中国畜牧兽医》 ›› 2018, Vol. 45 ›› Issue (8): 2293-2302.doi: 10.16431/j.cnki.1671-7236.2018.08.031

• 预防兽医 • 上一篇    下一篇

抗猪流行性腹泻病毒变异株卵黄抗体的制备、纯化及其活性影响因素的研究

雷丹1,2, 李安琪1,2, 罗素贤1,2, 叶昱1,2, 宋德平1,2, 唐玉新1,2   

  1. 1. 江西农业大学动物科学技术学院, 南昌 330045;
    2. 江西省畜禽疫病诊断与防控重点实验室, 南昌 330045
  • 收稿日期:2018-01-03 出版日期:2018-08-20 发布日期:2018-08-15
  • 通讯作者: 宋德平, 唐玉新 E-mail:sdpjxau@hotmail.com;tang53ster@gmail.com
  • 作者简介:雷丹(1992-),女,江西丰城人,硕士生,研究方向:动物传染病,E-mail:2313380557@qq.com;李安琪(1992-),女,江西萍乡人,硕士生,研究方向:动物传染病,E-mail:lianqi19920826@163.com
  • 基金资助:

    国家重点研发计划项目(2017YFD0500600);江西省教育厅科技计划项目(GJJ160399)

Preparation, Purification and Activity Factors Analysis of IgY Against Variant of Porcine Epidemic Diarrhea Virus

LEI Dan1,2, LI Anqi1,2, LUO Suxian1,2, YE Yu1,2, SONG Deping1,2, TANG Yuxin1,2   

  1. 1. College of Animal Science and Technology, Jiangxi Agricultural University, Nanchang 330045, China;
    2. Key Laboratory of Diagnosis and Control of Livestock and Poultry Diseases in Jiangxi Province, Nanchang 330045, China
  • Received:2018-01-03 Online:2018-08-20 Published:2018-08-15

摘要:

为制备抗猪流行性腹泻病毒(porcine epidemic diarrhea virus,PEDV)变异株卵黄抗体并探索卵黄抗体的纯化方法,本试验采用灭活的PEDV变异株CH/JX01全病毒免疫蛋鸡,收集免疫后所产的鸡蛋,分别用水稀释法、PEG-6000法和水稀释-硫酸铵二次沉淀法对鸡蛋中IgY进行提纯,然后运用间接ELISA和Western blotting方法鉴定和比较3种方法的提纯效果,获得可用于规模化生产的提纯方法;同时研究不同温度、pH条件下抗体活性,寻找对提纯IgY活性具有保护作用的物质。结果显示,本试验成功制备了抗PEDV变异株的卵黄抗体。浓度检测结果表明,水稀释法提取的IgY浓度最高,为6.204 mg/mL;PEG-6000方法提纯的IgY浓度次之,为4.673 mg/mL;水稀释-硫酸铵二次沉淀法IgY浓度最低,为3.359 mg/mL。间接ELISA检测结果表明,水稀释-硫酸铵二次沉淀法提纯的IgY效价最高,为1:12 800;其次是PEG-6000,为1:6 400;而水稀释法最低,为1:1 600。综合3种方法所提纯的卵黄抗体浓度与效价,发现水稀释-硫酸铵二次沉淀法的提纯效果优于其他两种方法。活性影响因素试验结果表明,提纯的IgY在37~60℃活性稳定;pH为4.0~11.0时具有良好的活性;在酸性条件下,20%硫糖铝对IgY活性具有较好的保护作用。本试验结果将为卵黄抗体的大规模生产运用与储存提供了有效的参考依据。

关键词: 卵黄抗体; 猪流行性腹泻病毒(PEDV); 水稀释法; PEG-6000法; 水稀释-硫酸铵二次沉淀法; 活性

Abstract:

The purpose of the experiment was to prepare the yolk antibody (IgY) against variant of porcine epidemic diarrhea virus (PEDV) and explore methods that could efficiently,easily prepare and purify the prepared IgY.In this study,hens were immunized by variant PEDV (CH/JX01) and then the eggs were collected.Three different methods,water dilution method,polyethylene glycol 6000 (PEG-6000) method and water dilution-ammonium sulfate secondary precipitation method were introduced to purify the IgY targeted to PEDV,and the extraction quantity were evaluated by indirect ELISA and Western blotting.The results showed that the concentration of IgY extracted by water dilution method was the highest,reaching 6.204 mg/mL,followed by PEG-6000 method,4.673 mg/mL,and the concentration of IgY by water dilution-ammonium sulfate secondary precipitation method was 3.359 mg/mL.Based on the results of ELISA,the titer of IgY targeted PEDV from water dilution-ammonium sulfate secondary precipitation method was the highest with a titer of 1:12 800,while the titers of IgY from PEG-6000 and water dilution methods were 1:6 400 and 1:1 600,respectively.Compared the three different methods of purified IgY concentration and titer,it concluded that the purification effect of the water dilution-ammonium sulfate secondary precipitation method was better than the other two methods.The activity factors analysis results indicated that IgY purified by the water dilution-ammonium sulfate secondary precipitation method was stable at 37 to 60℃ and had good activity at pH 4.0 to pH 11.0.Under acidic conditions,20% sucralfate had a good protective effect on IgY activity.The results would provide an effective reference for the large-scale production and storage of the yolk antibody in the future.

Key words: egg yolk antibody; porcine epidemic diarrhea virus (PEDV); water dilution method; PEG-6000 method; water dilution-ammonium sulfate secondary precipitation method; activity

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