Comparison and preliminary application of five extraction methods of egg yolk antibody

doi:10.14088/j.cnki.issn0439-8114.2020.15.021

Abstract

Abstract: In order to obtain a relatively good extraction process of egg yolk antibody, five methods including octanoic acid method, water dilution method, chloroform method, ammonium sulfate precipitation method and polyethylene glycol precipitation method were improved in this experiment. Expression of recombinant porcine pathogenic Escherichia coli toxin STxB（rSTxB） was induced by IPGT, and then the immunogen was prepared by emulsifying the rSTxB with the adjuvant 71VG, and the hens were immunized, IgY was extracted by modified polyethylene glycol method. The purity of the IgY antibody was detected by SDS-PAGE electrophoresis, and the immunoactivity and titers of yolk antibody and serum antibody titer were detected by Western blot. The results showed that the extraction efficiencies of octanoic acid method, water dilution method, chloroform method, ammonium sulfate precipitation method and polyethylene glycol precipitation method were 96.00,81.99,155.50,65.34,46.00 mg/mL,respectively, and the extraction purity of polyethylene glycol precipitation was the highest. The molecular weight of rSTxB protein was about 25 kDa, the antibody titer in serum was up to 64 000 times, and the highest dilution factor of yolk antibody was 16 000 times. For the comprehensive evaluation, the extraction method of polyethylene glycol precipitation was relatively superior, and the extracted yolk antibody showed good immunological activity.

Key words: egg yolk antibody, extraction process, polyethylene glycol precipitation, STxB protein, adjuvant

CLC Number:

S852.4⁺3

XIANG Dong-mei, YI Si-yu, TANG Qing-hai, WU Si-wen, WANG Shu-shu, LI Yan, LI Lu, HAN Tao-tao. Comparison and preliminary application of five extraction methods of egg yolk antibody[J]. HUBEI AGRICULTURAL SCIENCES, 2020, 59(15): 108-113.

References

[1] 李敏惠, 邹强, 杨淑霞, 等.鸡卵黄抗体IgY的分离纯化及鉴定[J].生物学杂志,2009,26(6):80-82.
[2] 马蕾, 姚刚, 杨光, 等. 微囊化与非微囊化胆囊收缩素卵黄抗体对小鼠促生长免疫效果的比较研究[J].中国畜牧兽医, 2013, 40(12):216-219.
[3] 张晓萍. 胆囊收缩素卵黄抗体的生物学活性及其作用机理初探[D]. 南京:南京农业大学, 2004.
[4] 邱涛涛, 徐振林, 甘庆庆, 等. 卵黄抗体体内代谢及中和毒素研究进展[J].食品科学,2019,40(5):303-308.
[5] WANG Q, HOU X J, CAI K, et al.Passive protection of purified yolk immunoglobulin administered against Shiga toxin 1 in mouse models[J]. Canedian journal of microbiology,2010,56(12):1003-1010.
[6] 王荣. 具有广谱抗ETEC腹泻功能鸡蛋的研制[D]. 武汉:华中农业大学, 2013.
[7] NERI P, TOKORO S, KOBAYASHI R, et al.Specific egg yolk immunoglobulin as a new preventive approach for Shiga-toxin-mediated diseases[J]. PLoS ONE, 2011, 6(10): e26526.
[8] MINE Y,KOVACS-NOLAN J.Chicken egg yolk antibodies as therapeutics in enteric infectious disease: A review[J]. Journal of medicinal food, 2002, 5(3):159-169.
[9] 吴琼. 抗仔猪腹泻卵黄抗体膏的研制及应用[D]. 辽宁大连:大连理工大学,2013.
[10] 吴义龙,费荣梅. 鸡卵黄抗体IgY研究进展[J].畜牧与兽医, 2013,45(3):94-98.
[11] 谢燕燕. 功能性卵黄粉的制备及其对仔猪生长性能的影响[D].福州:福建农林大学,2010.
[12] 阳元娥. 抗甲型流感病毒卵黄抗体的制备、活性评价及其应用研究[D]. 广州:广东工业大学,2016.
[13] 刘瑞生, 张洪波, 薛掌林, 等. 鸡新城疫卵黄抗体提取方法筛选研究[J]. 国外畜牧学—猪与禽, 2014, 34(6): 57-59.
[14] 吴博,王爽,樊兴冬,等. 不同方法提取卵黄抗体效果的比较[J]. 中国兽药杂志,2016, 46(6): 42-45.
[15] REN H, YANG W J, THIRUMALAI D, et al.A comparative evaluation of six principal IgY antibody extraction methods[J]. Altern Lab Anim, 2016, 44(1):11-20.
[16] 蒋兴龙, 江国永, 成国祥. 几种从鸡血浆中提取免疫球蛋白IgY方法的比较[J]. 动物源性蛋白与活性肽, 2015(1): 64-67.
[17] PAULY D, CHACANA P A, CALZADO E G, et al.IgY technology: Extraction of chicken antibodies from egg yolk by polyethylene glycol(PEG) precipitation[J].Journal of visualized experiments,2011(51):167-174.
[18] 马龙. 奶牛临床型乳房炎病原菌的分离鉴定及其卵黄抗体的制备[D]. 兰州:甘肃农业大学, 2009.
[19] 王辉龙, 周建武, 王中来, 等. 聚乙二醇一步沉淀提取卵黄抗体[J]. 福州大学学报(自然科学版), 2010, 29(5): 123-126.
[20] 朱旭国, 许甜甜, 杜宁. 聚乙二醇共沉复溶双水相法提纯氯过氧化物酶的条件研究[J]. 上海师范大学学报(自然科学版), 2010, 30(4): 415-420.
[21] 谭佩毅, 黄秀锦. 免疫卵黄抗体提取工艺的优化[J].食品与机械, 2012, 28(6):231-235.
[22] 索江华, 张粟. 传染性法式囊卵黄抗体不同提取工艺的比较[J]. 郑州牧业工程高等专科学校学报,2014, 34(2): 22-24.
[23] 任平国, 徐启红. 鸡卵黄抗体(IgY)的制备工艺研究[J]. 食品工业, 2011, 32(1):71-73.
[24] 任春芝. 鸡球虫多价卵黄抗体IgY的制备及其抗球虫效果评价[D]. 江苏扬州:扬州大学, 2011.
[25] 韩继刚, 康明, 刘平芳, 等. 利用聚乙二醇(PEG8000)纯化小球藻病毒 FJ-1[J]. 微生物学报, 2000, 40(5): 556-558.
[26] 高岭. 猪传染性胃肠炎病毒与猪流行性腹泻病毒卵黄抗体的制备及纯化工艺研究[D]. 河北保定:河北农业大学, 2014.
[27] 王金洛,徐福洲,杨兵,等. 仔猪腹泻多病原特异性卵黄抗体(IgY)治疗和预防剂的研究[A]. 中国畜牧兽医学会第一届中国养猪生产和疾病控制技术大会. 2005中国畜牧兽医学会学术年会论文集[C]. 北京:中国畜牧兽医学会,2005.805-808.