天然大豆球蛋白亚基的分离纯化

Isolation and purification of native glycinin subunits

利用变性剂脲和还原剂β-巯基乙醇解聚大豆球蛋白,在还原条件下通过DEAE琼脂糖凝胶F阴离子交换层析分离大豆球蛋白亚基,并对制备的亚基进行复性,从而建立了系统的分离纯化大豆球蛋白天然亚基的方法。对该方法分离亚基的产率和亚基的纯度进行评定,并利用大豆球蛋白免疫家兔制备的抗血清检测各种纯化亚基的免疫活性。结果表明:在还原条件下利用阴离子交换层析,可以有效地分离大豆球蛋白的碱性亚基和不同种类的酸性亚基。该方法的产率较高,以大豆球蛋白分离纯化碱性亚基及酸性亚基A1 a,A2,A3,A4的产率分别为23.93%,14.95%,14.33%,12.06%,7.12%。制备的碱性亚基和酸性亚基A1 a,A2,A3,A4的纯度分别为84.39%,90.48%,92.00%,65.91%,85.00%;纯化亚基能与抗大豆球蛋白血清特异性结合,具有免疫活性。

A novel systematic approach of isolation and purification of native Glycinin subunits was established. In this method, Glycinin was depolymerized by denaturant urea and reductant β - mercaptoethanol first, and Glycinin subunits were separated using anion exchange chromatography by DEAE -Sepharose F F under reduced condition, at last purified subunits were renatured. Purification yield of this method and purity of prepared subunits were determined, the immunological activity of the subunits was detected by antiserum produced by rabbit immunization experiments by using Glycinin. The results indicated that basic subunits and different kinds of acidic subunits could be isolated effectively from the Glycinin by anion exchange chromatography under reduced condition. Yield of the basic subunit and acidic subunit A1a, A2, A3, A4 were 23.93% , 14.95% , 14.33% , 12.06% and 7.12% respectively. And the purity of prepared basic subunit and acidic subunit A1a, A2, A3, A4 were 84.39% ,90.48% , 92% ,65.91% ,85 %, respectively. The subunits could specially bind to Glycinin antiserum and this proved that the subunits had immunological activity.