从基因工程菌分离纯化心钠素

Isolation and Purification of ANF from Genetic Engineering Bacteria

以高效表达的基因工程菌为材料，对以包涵体形式存在的目的产物——心钠素（ＡＮＦ）融合蛋白进行了酶切及分离纯化．先用ＴｒｉｔｏｎＸ－１００和低浓度尿素、盐酸胍洗涤，提高包涵体的纯度．再用高浓度的尿素处理，使蛋白变性溶解，脱氧胆酸钠对蛋白有明显的促溶作用．解决变性蛋白质去除变性剂时的聚合问题．在非变性条件下进行ＳｅｐｈａｄｅｘＧ－７５凝胶过滤，ＦＰＬＣ系统纯化，得到目的融合蛋白．用凝血酶对融合蛋白作特异性切割，释放出ＡＮＦ小肽，经ＦＰＬＣ分离鉴定，与标准ＡＮＦ有相同的ＴＲ值，说明其纯度已达均一．生物活性检测表明：有明显的放免活性和舒张血管。

The target products expressed as inclusion bodies were extracted and purified.The cultured cells with over produced hybrid protein were harvested and disrupted by lutrasonication.Highly purified inclusion bodies were obtained by washing with Trition X 100,and low concentration urea and guanidine hydrochloride.High concentration urea was used to resolve the pallet.It was distinct that sodium deoxycholate accelerated the process.Using Sephadex G 75 chromatography and Pharmasia FPLC system purified the fusion proteins.Meanwhile,the target fusion proteins had been showed by SDS PAGE.The purified proteins were treated with thrombin to release specifically active ANF.ANF identified by FPLC has the same T R value with standard ANF,which showed that the ANF was homogeneous.In analysis of radio immunoassay and biological activities,the recombinant ANF has demonstrated significant radioimmune activites vasorelaxation and diuretic activities.