离子交换层析分离纯化人结合珠蛋白的研究  

Isolation and purification of human haptoglobin by ion exchange chromatography

作　　者：闫晶晶[1,2] 赵雄[2] 马玉媛[2] 马小伟 吕茂民[2] 章金刚[1,2] 

机构地区：[1]广西医科大学,南宁530021 [2]军事医学科学院壁战输血研究所血液安全保障技术研究北京市重点实验室,北芽100850 [3]华兰生物工程股份有限公司,河南新乡453003

出　　处：《军事医学》2016年第7期569-572,592,共5页Military Medical Sciences

基　　金：国家863计划资助项目(2012AA021902)

摘　　要：目的探索应用离子交换层析从Cohn组分Ⅳ分离纯化人结合珠蛋白(haptoglobin,Hp)的方法,并对纯化产物进行初步鉴定及活性分析。方法 Cohn组分Ⅳ溶解液稀释后经利凡诺络合,收集上清并用50%硫酸铵进行沉淀,沉淀重溶后用Q Sepharose Fast Flow层析分离纯化得到目的蛋白。然后利用SDS-PAGE及免疫印迹对目的蛋白进行鉴定,并利用Native-PAGE方法测定纯化产物的活性。结果 Cohn组分Ⅳ经过利凡诺络合及盐析处理后能有效去除部分杂蛋白,并使目的蛋白富集。利用离子交换层析方法从Cohn组分Ⅳ中纯化得到的主要产物为Hp2-2,SDS-PAGE显示Hp纯度较好,免疫印迹分析进一步证明,利用该法成功分离得到目的蛋白Hp,且利用NativePAGE方法测得目的蛋白活性为2.8 U/ml。结论利用该法从Cohn组分Ⅳ中成功分离得到人Hp,方法简单,易于放大生产,具有较好应用前景。Objective To develop an effective process for isolating and purifying haptoglobin（ Hp） from Cohn fractionⅣ by a new ion exchange chromatography and to preliminarily identify and analyze the product of each purification step.Methods The fraction was first diluted and impurities were adsorbed with Rivanol. Then,the supernatant was treated with50% ammonium sulfate. Finally,the precipitate was redissolved,and Hp was purified further with Q Sepharose Fast Flow chromatography. Native-PAGE was used to measure the activity of the haptoglobin-bound hemoglobin,while SDS-PAGE analysis and immunoblot were used for identification of the target protein. Results After pretreatment,some of the impurities were removed from the Cohn fraction Ⅳ,and the target protein was enriched. In our case,the target protein was Hp and Hp2-2 was the main phenotype in the human plasma fraction Ⅳ. Target protein band and high purity were identified by SDS-PAGE. Immunoblot analysis further proved that this method could successfully isolate the target protein Hp,and the activity of 2. 8 U / ml was measured by Native-PAGE method. Conclusion Haptoglobin is successfully isolated from human Cohn fraction Ⅳ with this method. The purification process is simple and suitable for scale-up production with a good prospect.

关 键 词：结合珠蛋白 Cohn组分Ⅳ 纯化 鉴定 

分 类 号：O658.1[理学—分析化学] R945[理学—化学]