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

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。

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.