动物营养学报 >

2018 , Vol. 30 >Issue 1: 100 - 106

DOI: https://doi.org/10.3969/j.issn.1006-267x.2018.01.014

反刍动物营养 Ruminant nutrition

牛乳铁蛋白多克隆抗体的制备、纯化及定量检测

展开
  • 中国农业科学院北京畜牧兽医研究所, 动物营养学国家重点实验室, 北京 100193
王建(1990-),女,山东潍坊人,硕士研究生,从事反刍动物营养研究。E-mail:wangjian_1884@163.com

收稿日期: 2017-06-12

  网络出版日期: 2018-01-04

基金资助

国家重点研发计划（2016YFD0500507）；中国农业科学院科技创新工程（ASTIP-IAS07）；国家高层次人才特殊支持计划

收起

Preparation, Purification and Quantitative Identification of Bovine Lactoferrin Polyclonal Antibody

Expand
  • State Key Laboratory of Animal Nutrition, Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing 100193, China

Received date: 2017-06-12

  Online published: 2018-01-04

Fold

摘要

本试验旨在制备高纯度和特异性的牛乳铁蛋白多克隆抗体，为鉴定并定量检测牛奶样品中及奶牛乳腺组织中合成的乳铁蛋白提供试验材料。选用4只健康新西兰大白兔，初次免疫乳铁蛋白4周后进行加强免疫，每2周加强免疫1次，待血清达到抗体效价后，对兔进行心脏采血并分离血清，利用饱和硫酸铵法和蛋白A树脂纯化抗体，十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）和免疫印迹（Western-blot）法分别用于鉴定纯化后抗体的纯度和特异性。测定纯化后抗体效价，并绘制抗体抑制曲线。最后利用所得抗体对市售液态奶、奶牛乳腺组织匀浆液、奶粉样品中的乳铁蛋白进行定量检测。结果表明，本试验制备的兔抗牛乳铁蛋白多克隆抗体纯度较高、特异性较强，抗体浓度为11.02 mg/mL，效价达到1：128 000；采用该抗体测定的乳腺组织样品中乳铁蛋白浓度为16.13 μg/g，液态奶中接近于0 μg/g，奶粉中为5.28 μg/g。总之，本试验采用经过饱和硫酸铵法和蛋白A树脂2步纯化后得到了纯度较高、特异性较强的兔抗牛乳铁蛋白多克隆抗体，可以用于牛奶等产品的乳铁蛋白鉴定。

关键词： 乳铁蛋白; 多克隆抗体; 抗体纯化

本文引用格式

王建, 郝丽媛, 魏婧雅, 孙鹏 . 牛乳铁蛋白多克隆抗体的制备、纯化及定量检测[J]. 动物营养学报, 2018 , 30(1) : 100 -106 . DOI: 10.3969/j.issn.1006-267x.2018.01.014

Abstract

This study aimed at preparing polyclonal antibody against lactoferrin in milk with high purity and specificity to provide experimental materials for the identification and quantification of lactoferrin synthesized in milk and mammary gland. Four healthy New Zealand white rabbits were immunized with bovine lactoferrin and challenged again after 4 weeks. Thereafter, the rabbits were immunized once every 2 weeks until antibody titer was desirable. Then the blood was collected from the heart and the serum was obtained. The antibody was purified by the saturated ammonium sulfate method and protein A resin. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western-blot were used to identify the purity and specificity of the purified antibody. Antibody titer was determined and the curve of antibody inhibition was obtained. Finally, we used the obtained antibody to quantitatively determine lactoferrin in commercial liquid milk, milk powder and the homogenate of mammary gland tissue of dairy cows. The results showed that the obtained rabbit anti-bovine lactoferrin polyclonal antibody had high purity and specificity, the antibody concentration was 11.02 mg/mL, and the titer was 1:128 000; lactoferrin concentration in mammary gland tissue of dairy cows was 16.13 μg/g, that in commercial liquid milk was close to 0, and that in milk powder was 5.28 μg/g. In conclusion, lactoferrin polyclonal antibody with high purity and specificity was prepared by saturated ammonium sulfate method and protein A resin in the present study, which can be used to identify lactoferrin in products like milk.

Key words： lactoferrin; polyclonal antibody; purification of antibody

参考文献

[1] KIECKENS E,RYBARCZYK J,BARTH S A,et al.Effect of lactoferrin on release and bioactivity of Shiga toxins from different Escherichia coli O157:H7 strains[J].Veterinary Microbiology,2017,202:29-37.

[2] RYBARCZYK J,KIECKENS E,VANROMPAY D,et al.In vitro and in vivo studies on the antimicrobial effect of lactoferrin against Escherichia coli O157:H7[J].Veterinary Microbiology,2017,202:23-28.

[3] WAKABAYASHI H,ODA H,YAMAUCHI K,et al.Lactoferrin for prevention of common viral infections[J].Journal of Infection and Chemotherapy,2014,20(11):666-671.  

[4] YOU J M,SUN P,LI D F,et al..A novel method using immuno-affinity chromatography for isolating β-conglycinin from soybean proteins[J].Food Chemistry,2009,117(2):371-374.  

[5] 庞学燕,季昀,王洪荣,等.奶牛αs-酪蛋白多克隆抗体的制备、纯化及鉴定[J].动物营养学报,2012,24(11):2190-2194.

[6] 黑文静.大豆主要抗营养因子检测技术研究及初步应用[D].硕士学位论文.北京:中国农业大学,2012.

[7] MURATA M,WAKABAYASHI H,YAMAUCHI K,et al.Identification of milk proteins enhancing the antimicrobial activity of lactoferrin and lactoferricin[J].Journal of Dairy Science,2013,96(8):4891-4898.  

[8] CARVALHO C A M,SOUSA I P Jr,SILVA J L,et al.Inhibition of Mayaro virus infection by bovine lactoferrin[J].Virology,2014,452/453:297-302.

[9] ZHANG J S,LAI S Y,CAI Z X,et al.Determination of bovine lactoferrin in dairy products by ultra-high performance liquid chromatography-tandem mass spectrometry based on tryptic signature peptides employing an isotope-labeled winged peptide as internal standard[J].Analytica Chimica Acta,2014,829:33-39.

[10] HABING G,HARRIS K,SCHUENEMANN G M,et al.Lactoferrin reduces mortality in preweaned calves with diarrhea[J].Journal of Dairy Science,2017,100(5):3940-3948.  

[11] TSAKALI E,PETROTOS K,CHATZILAZAROU A,et al.Short communication:determination of lactoferrin in Feta cheese whey with reversed-phase high-performance liquid chromatography[J].Journal of Dairy Science,2014,97(8):4832-4837.  

[12] 龚广予,巫庆华,吴正钧,等.一种检测乳铁蛋白的方法——放射免疫扩散法[J].中国乳品工业,2001,29(3):27-29.

[13] 卢蓉蓉,许时婴,王璋.乳铁蛋白测定方法的比较[J].中国乳品工业,2002,30(5):123-125.

[14] 李姣,张施敬,佘之蕴,等.乳铁蛋白纯化和检测方法研究进展[J].广东化工,2014,41(14):123-124,130.

[15] 李震,陈永福.牛乳中β-酪蛋白检测的ELISA方法[J].山东农业大学学报:自然科学版,1999,30(4):451-452.

[16] 张涛,庞广昌.酶联免疫法快速测定原料乳中αs-酪蛋白质量浓度[J].中国乳品工业,2006,34(2):56-58.

[17] 张涛,庞广昌.酶联免疫法快速测定原料乳中κ-酪蛋白含量[J].食品工业科技,2006,27(11):179-181.

[18] 李岩.乳中乳铁蛋白检测方法的建立[D].硕士学位论文.郑州:河南农业大学,2008.

[19] 沈新义,耿培兰,叶伟民.人乳铁蛋白(Lactoferrin)的分离纯化和抗血清制备[J].上海免疫学杂志,1983,3(4):228-230.

[20] REDWAN E M,UVERSKY V N,EL-FAKHARANY E M,et al.Potential lactoferrin activity against pathogenic viruses[J].Comptes Rendus Biologies,2014,337(10):581-595.  

[21] CHEN K,ZHANG L,LI H,et al.Iron metabolism in infants:influence of bovine lactoferrin from iron-fortified formula[J].Nutrition,2015,31(2):304-309.  
Options
文章导航

/

〈 〉

    联系我们

  • 地址：北京市海淀区圆明园西路2号
    　　　中国农业大学动科动医大楼153室
  • 邮编：100193
  • 电话：010－62817823
  • 网址：http://www.chinajan.com
  • E-mail：cjan@chinajan.com
京公网安备 11010802041926号    京ICP备17050989号-2