从Cohn法组份Ⅳ中分离纯化人血浆蛋白C  

Isolation and purification of human protein C from Cohn Fraction Ⅳ

作　　者：王宗奎[1] 林方昭[1] 肖小璞[1] 谢亦武[1,2] 李长清[1] 

机构地区：[1]中国医学科学院北京协和医学院输血研究所,四川成都610081 [2]China Biologic Products,Inc.

出　　处：《中国输血杂志》2010年第5期355-360,共6页Chinese Journal of Blood Transfusion

基　　金：四川省科技支撑计划项目(2009SZ0217)

摘　　要：目的探索建立1种串联离子交换层析和固相化金属螯合亲和层析从Cohn法组份Ⅳ分离纯化蛋白C的方法 ,降低纯化蛋白C的成本,并实现血浆的综合利用。方法 4℃条件下,将Cohn法组份Ⅳ用PBS缓冲液抽提5h,离心(6500g)40min,上清液依次用0.45μm和0.22μm的滤膜过滤后,超滤透析;再通过串联离子交换层析和固相化金属螯合亲和层析,对蛋白C进行纯化。通过SDS-PAGE鉴定蛋白C的纯度;用考马斯亮蓝法通过紫外分光光度计测定总蛋白含量;通过WHO指定的发色底物法和凝固法(一期法)测定蛋白C活性。结果纯化的蛋白C在SDS-PAGE图谱上呈现1条62kD左右的蛋白带;经过离子交换层析,蛋白C的活性回收率为(48.19±9.22)%,纯化倍数为(3.75±0.56)倍;固相化金属螯合亲和层析后,蛋白C活性回收率为(59.61±5.59)%,纯化倍数为(36.79±3.72)倍;蛋白C总活性回收率为(28.77±9.45)%,纯化倍数为(136.64±6.82)倍,比活性为(11.70±0.89)IU/mg。结论串联离子交换层析和固相化金属螯合亲和层析可以从Cohn法组份Ⅳ中获得比活性较高的蛋白C浓缩物。Objective To establish a more inexpensive method for isolating and purifying protein C from Cohn Fraction Ⅳ by combining ion exchange chromatography and immobilized metal affinity chromatography, and to achieve an improved utilization of fresh frozen plasma.Methods Cohn Fraction Ⅳ was dissolved with phosphate buffered saline solution in a ratio of 1∶10 for 5 h at 4 ℃, and the suspension was centrifuged for 40 min（4 ℃,6 500 g）. The supernatant was loaded through 0.45μm and 0.22μm filters in turn, and then ultrafiltered and dialyzed. Protein C was purified further via ion exchange chromatography and immobilized metal affinity chromatography. Characterization of protein C was performed by SDS-PAGE. Total protein content was determined by Bradford under UV absorption at 595 nm. The activity of protein C was determined using chromogenic substrate assay and one-stage clotting assay referenced against the World Health Organization International Standard. Results The purified protein C concentrate showed only one protein band on gels by SDS-PAGE, of which the molecular weight was ca. 62kD.The ion exchange chromatography step achieved a（3.75±0.56） fold increase in specific activity over the initiating material at a （48.19±9.22）% yield; and the immobilized metal affinity chromatography step achieved an additional （36.79±3.72） fold purification and a （59.61±5.59） % yield. Thus, an overall （136.64±6.82） fold increase in purity and a （28.77±9.45） % yield was obtained; protein C ：C had a specific activity of （11.70±0.89） IU/mg protein. Conclusion The combination of ion exchange chromatography and immobilized metal affinity chromatography which is much less expensive could be an efficient method for protein C purification from Cohn Fraction Ⅳ.

关 键 词：Cohn法组份Ⅳ 蛋白C 分离 纯化 亲和层析 固相化金属螯合 比活性 

分 类 号：R392-33[医药卫生—免疫学] R457.14[医药卫生—基础医学]