基于串联亲和层析法纯化并鉴定猪初乳中SIgA

doi:10.11843/j.issn.0366-6964.2023.09.027

畜牧兽医学报 ›› 2023, Vol. 54 ›› Issue (9): 3884-3894.doi: 10.11843/j.issn.0366-6964.2023.09.027

基于串联亲和层析法纯化并鉴定猪初乳中SIgA

杨悦^1,2, 王锐³, 甘源^2,4, 郝飞^2,4, 谢星^2,4, 张磊^2,4, 邵国青^2,4, 孟庆国¹, 陈蓉^2,4*, 冯志新^1,2,4*

1. 南京师范大学海洋科学与工程学院, 南京 210023;
2. 江苏省农业科学院兽医研究所, 农业农村部兽用生物制品工程重点实验室, 南京 210014;
3. 南京农业大学生命科学院, 南京 210095;
4. 兽用生物制品(泰州)国泰技术创新中心, 泰州 225300

收稿日期:2023-02-13 发布日期:2023-09-22
通讯作者: 冯志新,主要从事动物支原体致病机制研究,E-mail:fzxjaas@163.com;陈蓉,主要从事动物支原体致病机制研究,E-mail:chenronggrape@163.com
作者简介:杨悦(1998-),女,江苏兴化人,硕士,主要从事动物支原体致病机制研究
基金资助:
国家重点研发计划政府间国际科技创新合作重点专项(2019YFE0107300);江苏省农业科技自主创新资金项目(CX(22)3029);国家自然基金面上项目(32273011)

Purification and Identification of SIgA in Porcine Colostrum based on Tandem Affinity Chromatography

YANG Yue^1,2, WANG Rui³, GAN Yuan^2,4, HAO Fei^2,4, XIE Xing^2,4, ZHANG Lei^2,4, SHAO Guoqing^2,4, MENG Qingguo¹, CHEN Rong^2,4*, FENG Zhixin^1,2,4*

1. School of Marine Science and Engineering, Nanjing Normal University, Nanjing 210023, China;
2. Key Laboratory of Veterinary Biological Products Engineering, Ministry of Agriculture and Rural Affairs, Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China;
3. School of Life Sciences, Nanjing Agricultural University, Nanjing 210095, China;
4. Guotai Technology Innovation Center of Veterinary Biological Products(Taizhou), Taizhou 225300, China

Received:2023-02-13 Published:2023-09-22

摘要/Abstract

摘要： 分泌型免疫球蛋白A(SIgA)是黏膜免疫的主要效应分子,在临床上常常作为疾病早期诊断的靶标,此外,基于SIgA的治疗性单抗和靶向SIgA产生的黏膜疫苗也越来越受到关注。分离并纯化出完整且具有活性的SIgA是产品研发的前提。为了提高猪初乳中SIgA的纯化效率,本研究采用串联亲和层析,强阴离子柱和精细分子筛层析进行纯化,从10 mL初乳中获得3 mg具有活性的SIgA。使用Western blot对纯化的SIgA进行鉴定,结果表明,纯化的SIgA与抗猪的重链蛋白单抗和抗SC单抗均具有良好的反应性。使用ELISA测试SIgA与抗猪的IgA重链单抗的反应性,结果发现使用本方法纯化的SIgA较原先方法(即硫酸铵沉淀,DEAE52弱阴离子层析,SephadexG-200凝胶层析和多次亲和层析的纯化方法)具有更高的反应性。最后使用质谱鉴定纯化的蛋白,结果表明为猪的SIgA。本研究为从猪初乳中纯化SIgA建立了一种高效的纯化方法,也为其它来源的SIgA的纯化提供参考。

关键词: 猪初乳, SIgA, 纯化, 鉴定

Abstract: SIgA is the primary effector molecule of mucosal immunity, and it is often used as a target for early diagnosis of diseases in clinic. In addition, more and more attention has been paid for therapeutic SIgA mAbs and mucosal vaccines targeting SIgA producing. Isolation and purification of intact and active SIgA is a prerequisite for product development. In order to improve the purification efficiency of SIgA from swine colostrum, in this study, a new way was established,including using tandem affinity chromatography, strong anion column and superpose 6 gel filtration. By using this way, 3 mg of active SIgA was abtained from 10 mL of colostrum. Western blot immunoblotting was performed with anti-pig IgA heavy chain MAb and anti-SC MAb, and the results showed that both of the MAbs had good reactivity with purified SIgA here. ELISA method was used to test the reactivity of SIgA purified here and anti-pig IgA heavy chain MAb, it showed that the purified SIgA in our study had higher reactivity than SIgA purified by the old method (ammonium sulphate precipitation, DEAE52 weak anion chromatography, SephadexG-200 gel chromatography and multiple Protein A affinity chromatography). Finally, purified SIgA was further checked by mass spectrometry. Our research established an efficient method for the purification of SIgA from swine colostrum, and also provided a reference for the purification of SIgA from other animals.

Key words: porcine colostrum, SIgA, purification, identification

中图分类号:

S852.4

杨悦, 王锐, 甘源, 郝飞, 谢星, 张磊, 邵国青, 孟庆国, 陈蓉, 冯志新. 基于串联亲和层析法纯化并鉴定猪初乳中SIgA[J]. 畜牧兽医学报, 2023, 54(9): 3884-3894.

YANG Yue, WANG Rui, GAN Yuan, HAO Fei, XIE Xing, ZHANG Lei, SHAO Guoqing, MENG Qingguo, CHEN Rong, FENG Zhixin. Purification and Identification of SIgA in Porcine Colostrum based on Tandem Affinity Chromatography[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(9): 3884-3894.

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基于串联亲和层析法纯化并鉴定猪初乳中SIgA

Purification and Identification of SIgA in Porcine Colostrum based on Tandem Affinity Chromatography

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